Bottlebrush polyethylene glycol nanocarriers

Abstract

Provided herein is a bottlebrush polyethylene glycol (PEG-BB) nanocarrier that can translocate across all barriers within the human airway surface.

Description

[0001] BOTTLEBRUSH POLYETHYLENE GLYCOL NANOCARRIERS

[0002] PRIORITY

[0003] This application claims the benefit of the filing date of U. S. application No. 63 / 626,879, filed January 30, 2024, the disclosure of which is incorporated by reference herein.

[0004] STATEMENT OF GOVERNMENT SUPPORT

[0005] This invention was made with government support under 1944625 awarded by the National Science Foundation. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] Pulmonary drug delivery1is used to the treatment of respiratory diseases, such as asthma2, chronic obstructive pulmonary disease (COPD)3, and pulmonary fibrosis4. However, the human airway surface has a multiple-layer structure5 7that presents multiscale barriers for efficient and localized drug delivery. Lining the airway surface is mucus, a viscoelastic and sticky hydrogel that traps essentially any inhaled particulates and pathogens8-10. The mucus is further separated from the epithelium by a periciliary layer, which provides a favorable environment for cilia beating and cell surface lubrication. Together with trapped objects, the mucus hydrogel is transported out of the lung by coordinated cilia beating. While needed to maintaining respiratory health, this mucociliary clearance also prevents the retention of drugs within the airway11,12. Furthermore, the epithelial cells are connected by cell junctions such as tight junctions, forming an integrated epithelial barrier that prevents drug carriers from traversing and reaching the underlying cells13-15. Thus, efficient pulmonary drug delivery requires carriers that can quickly penetrate mucus faster than its turnover rate, sneak through the periciliary layer, and then be internalized into and retained within epithelial cells.

[0008] Recent advances in drug delivery have demonstrated that coating sub-micrometer latex particles with bioinert polymers such as polyethylene glycol (PEG) promotes particle penetration through mucus16-18. However, as demonstrated by previous work5,19,20,7,8,21 24, the mucus hydrogel is highly heterogeneous with a wide distribution in the network mesh size from tens to hundreds of nanometers; thus, the PEGylated solid particles can still be physically trapped by local small meshes. Decreasing the particle size to -lOOnrn or less helps circumvent the physical confinement. However, such small nanoparticles have a large surface curvature that inevitably causes unreliable coating, such that the particles adhere to mucus through nonspecific biochemical binding25, a phenomenon commonly known as mucoadhesion26. In addition, it was recently discovered that the periciliary layer is not simply filled with low- viscosity physiological liquid; instead, it is gel-like with transmembrane mucins densely grafted to cilia and the cell surface, forming a brush-like gel with a mesh size of 20-40 nm5. This periciliary brush gel serves as a protective layer additional to mucus to prevent external objects from reaching the cell surface.

[0009] SUMMARY

[0010] Pulmonary drug delivery is needed to treat respiratory diseases. However, the human airway surface presents multiscale barriers for efficient drug delivery. Provided herein is a bottlebrush polyethylene glycol (PEG-BB) nanocarrier that can translocate across all barriers within the human airway surface. The PEG-BB comprises a long linear backbone densely grafted by many (-1,000) relatively low molecular weight (-1000 g / mol) PEG side chains; this results in a highly anisotropic, wormlike nanocarrier featuring a contour length of -250 nm, a cross-section of -20 nm, and a hydrodynamic diameter of -40 nm. Using the classic air-liquidinterface culture system to recapitulate essential biological features of the human airway surface, it was shown that PEG-BB rapidly penetrates through endogenous airway mucus and periciliary brush layer (mesh size of 20-40 nm) to be internalized by airway epithelial cells across the whole epithelium within -1 min. By quantifying the cellular uptake of polymeric carriers of various molecular architectures and manipulating cell proliferation and endocytosis pathways, it was shown that the translocation of PEG-BB across the epithelium is driven by bottlebrush-architecture-enhanced endocytosis. The results demonstrate that large, wormlike bottlebrush PEG polymers can be used as a novel carrier for pulmonary and mucosal drug delivery.

[0011] Some aspects provide a bottlebrush polyethylene glycol (PEG-BB) nanocarrier of polyethylene glycol (PEG) macromonomers forming a linear backbone grafted by PEG side chains, wherein the backbone has between about 750 to about 1200 side chains extending radially away from the backbone. In some aspects, the PEG macromonomers have a molecular weight of about 200 grams / mole (g / mol) to about 1500 g / mol, including PEG macromonomers having a molecular weight of about 400 grams / mole (g / mol) to about 1200 g / mol, PEG macromonomers having a molecular weight of about 1000 g / mol or PEG macromonomers having a molecular weight of about 950 g / mol. In some aspects, the PEG macromonomers are methacrylate-terminated. In some aspects, the grafting density of the PEG side chains is about 0.6 to abut four side chains per nanometer as measured by 'H NMR, including, grafting density of the PEG side chains is four side chains per nanometer (4 nm’1) as measured by1H NMR. In some aspects, there are no spacers in the backbone. In some aspects, PEG-BB has a contour length of about 150-350 nm, a cross-section of about 15-30 nm, and a hydrodynamic diameter of about 30-50 nm, including, a contour length of about 250 nm, a cross-section of about 20 nm, and a hydrodynamic diameter of about 40 nm. Some aspects further comprise one or more therapeutic, prophylactic, or diagnostic agents.

[0012] Some aspects provide a pharmaceutical composition comprising the nanocarrier provided herein and one or more pharmaceutically acceptable carriers.

[0013] Some aspects provide a method of administering one or more therapeutic, prophylactic, and / or diagnostic agents to a subject in need thereof, the method comprising administering an effective amount of the nanocarrier disclosed herein.

[0014] Some aspects provide a method of administering one or more therapeutic, prophylactic, and / or diagnostic agents to airway epithelium cells of a subject in need thereof, the method comprising administering an effective amount of the nanocarrier of provided herein. In some aspects, the nanocarrier is administered enterally, parenterally, or topically. In some aspects, the nanocarrier is administered to a pulmonary tract.

[0015] Some aspects provide a method to treat a respiratory or mucus disease or disorder comprising administering the nanocarrier provided herein.

[0016] DRAWINGS

[0017] The features of the present disclosure are set forth with particularity in the appended claims. Abetter understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0018] FIGs 1A-1G. Design and synthesis of grafted PEG polymeric nanocarriers for pulmonary delivery, (a) The human airway epithelial barrier consists of three distinct layers: (i) mucus hydrogel, (ii) periciliary layer, and (iii) a pseudostratified epithelium in which the cells are connected by cell junctions, (b) The mucus hydrogel has a mesh size in the range of 10-100 nm. In the periciliary layer, transmembrane mucins are tethered to cilia and the epithelial surface to form a brush-like layer with a mesh size in the range of 20-40 nm. (c) The grafted PEG macromolecule is a random copolymer consisting of side chains (PEG methacrylate of 950 g / mol), spacers (2-methoxyethyl acrylate), and fluorescent labels (fluorescein O-methacrylate). The design parameter space is [rise, s, f\, in which nscis the number of side chains, 5 is the spacer / side chain ratio, and / is the molar fraction of fluorescein, (d) Upper: A densely grafted bottlebrush PEG (PEG-BB) with 950 side chains and no spacers. The polymer is a worm-like molecule with a cross-section size of ~20 nm and a contour length of -250 nm. Lower: A loosely grafted PEG (PEG-LG) with approximately 750 side chains, in which two neighboring side chains are separated by 5 spacer monomers on average. The polymer is a comb-like molecule with a cross-section size of -6 nm and a contour length of -1000 nm (Example II). (e) GPC profiles of PEG-BB and PEG-LG nearly overlap, indicating the two molecules have similar hydrodynamic diameter. The poly dispersity index for both polymers is the same of 1.45. (f) Dynamic light scattering (DLS) of PEG-BB and PEG-LG at room temperature, (g) The Z-average hydrodynamic diameter is about 37 nm for PEG-BB and about 32 nm for PEG-LG. Moreover, their sizes are nearly independent of temperature from 20 °C to 45 °C. Error bar is smaller than the symbol size («=3).

[0019] FIGS. 2A-2G. Uptake of PEG-BB by well-differentiated HBECs across the mucus barrier and the periciliary brush layer from the apical side, (a, b) Immediate uptake of PEG-BB by HBECs. (a) In this study, 10 pl of 1 mg / ml PEG-BB was added to the apical side of a well- differentiated HBEC culture. After incubation for approximately 1 min, PEG-BB was washed away using DPBS, and then the cells were imaged at different depths into the HBEC layer, (b) Fluorescence confocal images showing the uptake of PEG-BB of cells (i) on the apical side, (ii) in the middle, and (iii) on the basal side of the HBEC layer (red dashed lines at timepoint (T) in (a)), (c-g) Long-term uptake of PEG-BB by HBECs with intact mucus, (c) In this study, 10 pl of 1 mg / ml PEG-BB was added to the apical side to incubate HBECs overnight. Cells are imaged with intact mucus on the next day. 70 kDa Texas Red™ dextran was added to the apical side to mark the periciliary brush layer and to outline the upper boundary of the epithelium. Cells are imaged both along XY and XZ cross-sections, (d) XY images of the cells on apical side (dashed line at timepoint (T) in (c). (e) XY images of the epithelial layer taken at the level of the periciliary brush layer (dashed line at timepoint @ in (c)). (f) XZ profile of the epithelial layer, taken at timepoint @ in (c), showing the uptake of 1 MDa PEG-BB (green) but not 70 kDa dextran (red) by HBECs across the whole epithelial barrier, (g) PEG-BB accumulates in the cytoplasm but not the nuclei of HBECs. Arrowhead: cilia; white arrowhead: nucleus.

[0020] FIGS. 3A-3G. Uptake of PEG-BB from basal side and PEG-LG from apical side by HBECs. (a-d) Uptake of PEG-BB by HBECs from basal side, (a) PEG-BB was added to the basal side at the concentration of 100 mg / ml to incubated HBECs overnight. On the next day, was changed to remove free PEG-BB and imaging was performed at the apical focal plane. 70 kDa Texas Red™ dextran was added to the apical side to outline the periciliary brush layer. Imaging of the cells was performed along both XY and XZ profiles, (b) A representative XY fluorescence image of HBECs near the apical side (dashed line at timepoint (T) in (a)), (c) XZ profile of the epithelial layer, (d) XY image of HBECs at the level of the periciliary layer (dashed line at timepoint @ in (a)), (e, f) Uptake of PEG-LG by HBECs from the apical side. 10 pl of 1 mg / ml PEG-LG was added to the apical chamber. 70 kDa Texas Red™ dextran was added to the apical side to outline the periciliary brush layer. Imaging was performed at the apical focal plane along both XY and XZ profiles, (e) A representative XY fluorescence image of HBECs on the apical side, (f) XZ profile of the distribution of PEG-LG in the HBEC epithelial layer, (g) Quantitative comparison for the uptake of PEG-BB and PEG-LG by HBECs from basal and apical sides. Statistical analysis was performed using one-way ANOVA. n.s., not significant; **, / ?<0.01; ***, / ?<0.001. n=5 donors.

[0021] FIGS. 4A-4F. Uptake and retention of PEG-BB molecules within single NIH-3T3 fibroblasts, (a) Uptake of PEG-BB by NIH-3T3 fibroblasts after incubation overnight with 100 pg / mL PEG-BB in the medium on Days 1, 2, and 3. (b) Images showing intracellular PEG-BB fluorescence after inhibiting mitosis by 20 ng / mL colchicine before overnight incubation with 100 pg / ml PEG-BB in the medium. Cell nuclei were stained with 20 pg / mL Hoechst 33342 for 5 min at 37 °C. (c) Fluorescent cell fraction (Fc) with and without 20 ng / mL colchicine treatment. Fcis calculated by the number of fluorescent cells divided by the total cell count. Cells with any fluorescence regardless of the fluorescence area or intensity are counted. n=5 wells, (d) Relative cell count (CQ with and without colchicine treatment. Cr is defined as the ratio of cell counts normalized to the initial cell number on Day 1. n=5 wells. These results show that the concentration of colchicine was adequate to inhibit mitosis without compromising cell viability, (e) Fluorescence index (P) with or without 20 ng / mL colchicine treatment. P was defined as the product of Fcand Cr. P=Fc*Cr. n=5 wells, (f) PEG-BB fluorescent area fraction (Fa) with and without colchicine treatment. Fawas derived by the fluorescent area (A ) divided by the cytoplasm area. The cytoplasm area was the cell area (Ac) minus the nuclear area (An). The results demonstrate that PEG-BB fluorescence was relatively uniformly distributed in the cytoplasm and unchanged over 3 days after inhibiting mitosis. Statistical analysis was performed using one-way ANOVA. n.s., not significant; *****, p<0.00001. «=100 cells.

[0022] FIGS. 5A-5J. Cellular uptake of PEG-BB was driven by bottlebrush-architecture enhanced endocytosis. (a-c) Uptake of polymers with different molecular architectures by NIH- 3T3 fibroblasts: (a) 2 MDa dextran, a randomly branched inert molecule, (b) 70 kDa dextran, and (c) 1 MDa PEG-LG. All polymers were added to the medium at the same concentration of 100 pg / ml for overnight incubation, (d-f) Uptake of PEG-BB by NIH-3T3 fibroblasts was mediated by endocytosis. Representative images of NIH-3T3 fibroblasts treated for 4 hr with (d) 5 pg / ml chlorpromazine in the medium, (e) 5 pg / ml nystatin in the medium, and (f) 0.1 pg / ml wortmannin in the medium. Then, the cells are washed with prewarmed culture medium and incubated overnight with 100 pg / ml PEG-BB in the culture medium. Pre-wash: cells were imaged right after incubation without replacing cell culture medium. Post-wash: cells were imaged after being washed with fresh culture medium, (g-j) Uptake of PEG-BB by HBECs was significantly reduced after applying endocytosis inhibitors. Representative images of HBECs after treatment with (g) 0.1 pg / ml wortmannin in the medium, (h) 5 pg / ml chlorpromazine in the medium, (i) 5 pg / ml nystatin in the medium for 4 hr followed by adding 10 pl of 1 mg / ml PEG-BB from the apical side or adding PEG-BB to the basal side achieving a concentration of 100 pg / ml in the medium. Images were for cells right below the apical surface, (j) Fluorescence fraction of PEG-BB in HBECs after treatment with endocytosis inhibitors. n=5 donors, n.s., not significant; *****, / ?<0.00001. (k) A schematic illustrating that the bottlebrush architecture of PEG-BB promotes cellular uptake via endocytosis.

[0023] FIGS. S1A-S1B. Cross-section of a bottlebrush polymer in solution, (a) An unperturbed bottlebrush polymer with thickness R expected to be larger than the size of the free side chain. The correlation length ^(r) increases with the distance r from the bottlebrush backbone, (b) Mesh size profile for an unperturbed bottlebrush polymer. Yet, at a very high grafting density with the distance 1 between two neighboring grafting sites much smaller than the Kuhn monomer size b, the side chains form an exclusion zone (dashed line), as illustrated by the shadowed light blue circle in (a). Logarithmic scales.

[0024] FIG. S2. 'H NMR spectra of densely grafted bottlebrush PEG (PEG-BB). The conversion of PEG is [1 — (1 - 17.13) x 3] x 100% = 82.5% . The final degree of polymerization (DP) for PEG-BB is 1200 X 82.5% = 990. The feeding molar ratio between PEG and fluorescein o-acrylate is 100: 1.

[0025] FIG. S3. 'H NMR spectra of pure loosely grafted PEG (PEG-LG). The final molar ratio between PEG and MEA can be calculated in two ways. One is MPEG : MMEA = [Area(e+f) / 2- Area(a) / 3] : [Area(a) / 3], which is [11.96 / 2-3 / 3] : [3 / 3] = 5 : 1. The other way is MPEG : MMEA = [Area(c+d) / 3-Area(a) / 3] : [Area(a) / 3], which is [18.06 / 3-1]: ! = 5 : 1. The feeding ratio between PEG, MEA, and Flu is 5: 1 :0.06.

[0026] FIGS. S4A-S4C. Uptake of polymers with different molecular architectures by NIH- 3T3 fibroblasts. Representative pre- and post-wash images of NIH-3T3 fibroblasts after overnight incubation with (a) 2 MDa dextran, a randomly branched inert molecule, (b) 70 kDa dextran, and (c) 1 MDa PEG-LG. All polymers were added to the medium at the same concentration of 100 pg / ml. Pre-wash: cells were imaged right after incubation without replacing the cell culture medium. Post-wash: cells were imaged after being washed with fresh culture medium to remove free extracellular molecules. After overnight incubation, FITC- labeled 2 MDa dextran was not taken up by NIH-3T3 cells, rendering the cell contour circumscribed. 70 kDa dextran was negligibly taken up into NIH-3T3 cells as minimal perinuclear dots. PEG-LG, which has a comparable molecular weight and hydrodynamic size to PEG-BB, was taken up into NIH-3T3 cells slightly higher than 70 kDa dextran, showing as perinuclear fluorescence dots. However, the uptake of PEG-LG was significantly less than PEG-BB.

[0027] FIGS. S5A-S5C. Cell uptake of PEG-BB was inhibited by endocytosis inhibitors. Representative images ofNIH-3T3 cells treated with (a) 0.1 pg / ml wortmannin in the medium, (b) 5 pg / ml chlorpromazine in the medium, and (c) 5 pg / ml nystatin in the medium for 4 hours. Then, cells were washed with prewarmed culture medium and incubated overnight with 100 pg / ml PEG-BB in the culture medium. Pre-wash: cells were imaged right after incubation without replacing the culture medium. Post-wash: cells were imaged after being washed with fresh culture medium to remove free extracellular PEG-BB. After being treated with wortmannin, chlorpromazine, and nystatin to inhibit endocytosis, almost all PEG-BB was unabsorbed and remains extracellular, outlining cell contour in the pre-wash images. After washing, minimal PEG-BB remains intracellular and presents as perinuclear fluorescence dots in the post-wash images.

[0028] DESCRIPTION

[0029] The following descriptions and examples illustrate embodiments of the present disclosure in detail. Although the present disclosure has been described in some details by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims.

[0030] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0031] Although various features of the disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment. It is to be understood that the present disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there can be variations and modifications of the present disclosure, which can be encompassed within its scope. All patent filings, websites, other publications, accession numbers and the like cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated.

[0032] Any feature, step, element, embodiment, or aspect of the disclosure can be used in combination with any other unless specifically indicated otherwise.

[0033] Definitions

[0034] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated cases, e.g., to any commonly owned patent or application. Any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0035] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0036] In this application, the use of “or” means “and / or” unless stated otherwise. The terms “and / or” and “any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C”. The term “or” can be used conjunctively or disjunctively unless the context specifically refers to a disjunctive use.

[0037] Furthermore, the use of the term “including” as well as other forms, such as “include”, “includes” and “included”, is not limiting.

[0038] Reference in the specification to “some embodiments”, “an embodiment”, “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

[0039] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0040] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, z.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, the amount “about 10” includes 10 and any amounts from 9 to 11. In yet another example, the term “about” in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. Alternatively, particularly with respect to biological systems or processes, the term “about” can mean within an order of magnitude, such as within 5-fold, and within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0041] The term “treating” or preventing a disease, disorder or condition from occurring in an subject which may be predisposed to the disease, disorder and / or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and / or condition. Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0042] The term “therapeutic agent” refers to an agent that can be administered to prevent or treat a disease or disorder. Therapeutic agents can be a nucleic acid, a nucleic acid analog, a small molecule, a peptidomimetic, a protein, peptide, carbohydrate or sugar, lipid, or surfactant, or a combination thereof. “Nucleic Acid,” as used herein, refers to DNA, RNA, and nucleic acid molecules modified to increase stability for a variety of therapeutic purposes. One example is a gene encoding the human cystic fibrosis transmembrane conductance regulator (CFTR) protein, analogs and variants thereof, that can be expressed in CF individuals to correct at least in part some of the symptom’s characteristic of CF. This also include molecules such as DNA fragments including regions for introducing corrections or modifications into the gene, such as triple helix forming DNA, that can be used to correct the endogenous CF gene in at least some of the CF patient's genes. It should be noted that this term is not limited to CFTR genes but applied to every genetic material that can be used to treat, diagnose or cure disease.

[0043] The term “therapeutic agent” refers to an agent that can be administered to prevent or treat a disease or disorder. Therapeutic agents can be a nucleic acid, a nucleic acid analog, a small molecule, a peptidomimetic, a protein, peptide, carbohydrate or sugar, lipid, or surfactant, or a combination thereof.

[0044] The term “therapeutically effective amount” refers to an amount of the therapeutic agent that, when incorporated into and / or onto particles described herein, produces some desired effect at a reasonable benefit / risk ratio applicable to any medical treatment. The effective amount may vary depending on such factors as the disease or condition being treated, the particular targeted constructs being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation.

[0045] “Pulmonary administration,” as used herein, refers to administration of a pharmaceutical formulation containing an active agent into the lungs by inhalation. As used herein, the term “inhalation” refers to intake of air to the alveoli. The intake of air can occur through the mouth or nose. The intake of air can occur by self-administration of a formulation while inhaling, or by administration via a respirator to a patient on a respirator.

[0046] “Pharmaceutically acceptable,” as used herein, refers to compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio, in accordance with the guidelines of agencies such as the Food and Drug Administration.

[0047] The term "subject" includes animals, including both vertebrates and invertebrates, including, without limitation, invertebrates such as arthropods, mollusks, annelids, and cnidarians; and vertebrates such as amphibians, including frogs, salamanders, and caecillians; reptiles, including lizards, snakes, turtles, crocodiles, and alligators; fish; mammals, including human and non-human mammals such as non-human primates, including chimpanzees and other apes and monkey species; laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas; domestic animals such as dogs and cats; farm animals such as sheep, goats, pigs, horses and cows; and birds such as domestic, wild and game birds, including chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. In some cases, the disclosed methods find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, primates, and transgenic animals.

[0048] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub combination was individually and explicitly disclosed herein.

[0049] PEG-BB Nanocarrier

[0050] Polyethylene glycol (PEG) is a polyether compound derived from petroleum with many applications, from industrial manufacturing to medicine. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular weight. The structure of PEG is commonly expressed as H-(O-CH2-CH2)n-OH.

[0051] Representative PEG molecular weights for use in PEG-BB nanocarriers include about 500 grams / mole (g / mol) to about 10,000 g / mol, including about 500 g / mol, about 600 g / mol, about 800 g / mol, about 1000 g / mol, about 1500 g / mol, about 2000 g / mol, about 3000 g / mol, about 4000 g / mol, about 5000 g / mol, about 6000 g / mol, about 7000 g / mol, about 8000 g / mol, or about 10000 g / mol. In some embodiments, the PEG has a molecular weight of about 500 grams / mole (g / mol) to about 5000 g / mol, including about 1000 g / mol.

[0052] In some embodiments, the grafting density of the PEG side chains is about 0.6 to about 4 side chains per nanometer as measured byJH NMR, including 0.6 or 4 side chains per nanometer (4 nm’1) as measured by1H NMR. In some embodiments, the PEG-BB has a contour length of about 150-350 nm, a cross-section of about 15-30 nm, and a hydrodynamic diameter of about 30-50 nm, including a contour length of about 250 nm, a cross-section of about 20 nm, and a hydrodynamic diameter of about 40 nm.

[0053] The PEG-BB can be coated with or contain one or more surface altering agents or materials. “Surface-alternating agents”, as used herein refers to an agent or material which modifies one or more properties of the particles for the surface, including, but not limited to, hydrophilicity (e.g., makes the particles more or less hydrophilic), surface charge (e.g., makes the surface neutral or near neutral or more negative or positive), and / or enhances transport in or through bodily fluids and / or tissues, such as mucus. In some embodiments, the surfacealternating material provides a direct therapeutic effect, such as reducing inflammation.

[0054] Examples of the surface-altering agents include, but are not limited to, proteins, including anionic proteins (e.g., albumin), surfactants, sugars or sugar derivatives (e.g., cyclodextrin), therapeutics agents, and polymers. Polymers include heparin, polyethylene glycol (“PEG”) and poloxomers (polyethylene oxide block copolymers).

[0055] Examples of surfactants include, but are not limited to, L-a-phosphatidylcholine (PC), 1,2-dipalmitoylphosphatidy choline (DPPC), oleic acid, sorbitan trioleate, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monooleate, natural lecithin, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, block copolymers of oxyethylene and oxypropylene, synthetic lecithin, diethylene glycol dioleate, tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glyceryl monooleate, glyceryl monostearate, glyceryl monoricinoleate, cetyl alcohol, stearyl alcohol, polyethylene glycol 400, cetyl pyridinium chloride, benzalkonium chloride, olive oil, glyceryl monolaurate, corn oil, cotton seed oil, and sunflower seed oil, lecithin, oleic acid, and sorbitan trioleate.

[0056] Respiratory and / or Mucus Diseases

[0057] In the respiratory system, mucus is part of the airway surface liquid (ASL), also known as epithelial lining fluid (ELF), that lines most of the respiratory tract. The airway surface liquid consists of a sol layer termed the periciliary liquid layer and an overlying gel layer termed the mucus layer. The periciliary liquid layer is so named as it surrounds the cilia and lies on top of the surface epithelium. The periciliary liquid layer surrounding the cilia consists of a gel meshwork of cell-tethered mucins and polysaccharides. The mucus blanket aids in the protection of the lungs by trapping foreign particles before they can enter them, in particular through the nose during normal breathing. Mucus is made up of a fluid component of around 95% water, the mucin secretions from the goblet cells, and the submucosal glands (2-3% glycoproteins), proteoglycans (0.1- 0.5%), lipids (0.3-0.5%), proteins, and DNA. The major mucins secreted - MUC5AC and MUC5B - are large polymers that give the mucus its rheologic or viscoelastic properties. MUC5AC is the main gel-forming mucin secreted by goblet cells, in the form of threads and thin sheets. MUC5B is a polymeric protein secreted from submucosal glands and some goblet cells, and this is in the form of strands.

[0058] In the airways — the trachea, bronchi, and bronchioles — the lining of mucus is produced by specialized airway epithelial cells called goblet cells, and submucosal glands. Small particles such as dust, particulate pollutants, and allergens, as well as infectious agents and bacteria are caught in the viscous nasal or airway mucus and prevented from entering the system. This process, together with the continual movement of the cilia on the respiratory epithelium toward the oropharynx (mucociliary clearance), helps prevent foreign objects from entering the lungs during breathing. This explains why coughing often occurs in those who smoke cigarettes. The body's natural reaction is to increase mucus production. In addition, mucus aids in moisturizing the inhaled air and prevents tissues such as the nasal and airway epithelia from drying out.

[0059] Mucus is produced continuously in the respiratory tract. Mucociliary action carries it down from the nasal passages and up from the rest of the tract to the pharynx, with most of it being swallowed subconsciously. Sometimes in times of respiratory illness or inflammation, mucus can become thickened with cell debris, bacteria, and inflammatory cells. It is then known as phlegm which may be coughed up as sputum to clear the airway.

[0060] Increased mucus production in the upper respiratory tract is a symptom of many common ailments, such as the common cold, and influenza.

[0061] In the lower respiratory tract impaired mucociliary clearance due to conditions such as primary ciliary dyskinesia may result in mucus accumulation in the bronchi. The dysregulation of mucus homeostasis is the fundamental characteristic of cystic fibrosis, an inherited disease caused by mutations in the CFTR gene, which encodes a chloride channel. This defect leads to the altered electrolyte composition of mucus, which triggers its hyperabsorption and dehydration. Such low-volume, viscous, acidic mucus has a reduced antimicrobial function, which facilitates bacterial colonization. The thinning of the mucus layer ultimately affects the periciliary liquid layer, which becomes dehydrated, compromising ciliary function, and impairing mucociliary clearance. In the lower respiratory tract excessive mucus production in the bronchi and bronchioles is known as mucus hypersecretion. Chronic mucus hypersecretion results in the chronic productive cough of chronic bronchitis and is generally synonymous with this. Excessive mucus can narrow the airways, limit airflow, and accelerate a decline in lung function.

[0062] Respiratory diseases, or lung diseases, are pathological conditions affecting the organs and tissues that make gas exchange difficult in air-breathing animals. They include conditions of the respiratory tract including the trachea, bronchi, bronchioles, alveoli, pleurae, pleural cavity, the nerves and muscles of respiration. Respiratory diseases range from mild and selflimiting, such as the common cold, influenza, and pharyngitis to life-threatening diseases such as bacterial pneumonia, pulmonary embolism, tuberculosis, acute asthma, lung cancer, and severe acute respiratory syndromes, such as COVID-19. Respiratory diseases can be classified in many different ways, including by the organ or tissue involved, by the type and pattern of associated signs and symptoms, or by the cause of the disease.

[0063] Asthma, chronic bronchitis, bronchiectasis and chronic obstructive pulmonary disease (COPD) are all obstructive lung diseases characterized by airway obstruction. This limits the amount of air that is able to enter alveoli because of constriction of the bronchial tree, due to inflammation. Obstructive lung diseases are often identified because of symptoms and diagnosed with pulmonary function tests such as spirometry. Many obstructive lung diseases are managed by avoiding triggers (such as dust mites or smoking), with symptom control such as bronchodilators, and with suppression of inflammation (such as through corticosteroids).

[0064] Restrictive lung diseases are a category of respiratory disease characterized by a loss of lung compliance, causing incomplete lung expansion and increased lung stiffness, such as in infants with respiratory distress syndrome. Restrictive lung diseases can be divided into two categories: those caused by intrinsic factors and those caused by extrinsic factors. Restrictive lung diseases yielding from intrinsic factors occur within the lungs themselves, such as tissue death due to inflammation or toxins. Conversely, restrictive lung diseases caused by extrinsic factors result from conditions originating from outside the lungs such as neuromuscular dysfunction.

[0065] Chronic respiratory diseases are long-term diseases of the airways and other structures of the lung. They are characterized by a high inflammatory cell recruitment (neutrophil) and / or destructive cycle of infection, (e.g., mediated by Pseudomonas aeruginosa). Some of the most common are asthma, chronic obstructive pulmonary disease, and acute respiratory distress syndrome. Infections can affect any part of the respiratory system. They are traditionally divided into upper respiratory tract infections and lower respiratory tract infections. The upper airway is defined as all the structures connecting the glottis to the mouth and nose. The most common upper respiratory tract infection is the common cold. However, infections of specific organs of the upper respiratory tract such as sinusitis, tonsillitis, epiglottitis, croup, otitis media, pharyngitis and laryngitis are also considered upper respiratory tract infections.

[0066] The most common lower respiratory tract infection is pneumonia, an infection of the lungs which is usually caused by bacteria, particularly Streptococcus pneumoniae in Western countries. Worldwide, tuberculosis is an important cause of pneumonia. Other pathogens such as viruses and fungi can cause pneumonia, for example severe acute respiratory syndrome, COVID-19 and pneumocystis pneumonia. Pneumonia may develop complications such as a lung abscess, a round cavity in the lung caused by the infection.

[0067] Primary ciliary dyskinesia is a genetic disorder causing the cilia to not move in a coordinated manner. This causes chronic respiratory infections, cough, and nasal congestion. This can lead to bronchiectasis, which can cause life-threatening breathing issues.

[0068] Malignant tumors of the respiratory system, particularly primary carcinomas of the lung, are a major health problem responsible for 15% of all cancer diagnoses and 30% of all cancer deaths. The major histological types of respiratory system cancer include small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, large cell lung carcinoma, other lung cancers (carcinoid, Kaposi's sarcoma, melanoma), lymphoma, head and neck cancer and pleural mesothelioma.

[0069] Compounds / Drugs / Administration

[0070] Therapeutic Agents

[0071] In some embodiments, the PEG-BB have encapsulated therein, dispersed therein, and / or covalently or non-covalently associated with the surface one or more therapeutic agents. The therapeutic agent can be a small molecule, protein, polysaccharide or saccharide, nucleic acid molecule and / or lipid.

[0072] Small Molecule Therapeutic Agents

[0073] Exemplary classes of small molecule therapeutic agents include, but are not limited to, analgesics, anti-inflammatory drugs, antipyretics, antidepressants, antiepileptics, antiopsychotic agents, neuroprotective agents, anti-proliferatives, such as anti-cancer agent, anti-infectious agents, such as antibacterial agents and antifungal agents, antihistamines, antimigraine drugs, antimuscarinics, anxioltyics, sedatives, hypnotics, antipsychotics, bronchodilators, anti-asthma drugs, cardiovascular drugs, corticosteroids, dopaminergics, electrolytes, gastro-intestinal drugs, muscle relaxants, nutritional agents, vitamins, parasympathomimetics, stimulants, anorectics and anti-narcoleptics.

[0074] Nucleic Acids

[0075] In some embodiments, the agent is one or more nucleic acids. The nucleic acid can alter, correct, or replace an endogenous nucleic acid sequence. The nucleic acid is used to treat cancers, correct defects in genes in other pulmonary diseases and metabolic diseases affecting lung function, genes such as those for the treatment of Parkinson’s and ALS where the genes reach the brain through nasal delivery.

[0076] Gene therapy is a technique for correcting defective genes responsible for disease development. Researchers may use one of several approaches for correcting faulty genes:

[0077] A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common.

[0078] An abnormal gene could be swapped for a normal gene through homologous recombination.

[0079] The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function.

[0080] The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.

[0081] The nucleic acid can be a DNA, RNA, a chemically modified nucleic acid, or combinations thereof. For example, methods for increasing stability of nucleic acid half-life and resistance to enzymatic cleavage are known in the art and can include one or more modifications or substitutions to the nucleobases, sugars, or linkages of the polynucleotide. The nucleic acid can be custom synthesized to contain properties that are tailored to fit a desired use. Common modifications include but are not limited to use of locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), morpholinos, peptide nucleic acids (PNA), phosphorothioate linkages, phosphonoacetate linkages, propyne analogs, 2'-O-methyl RNA, 5-Me-dC, 2'-5' linked phosphodiester linage, Chimeric Linkages (Mixed phosphorothioate and phosphodiester linkages and modifications), conjugation with lipid and peptides, and combinations thereof.

[0082] In some embodiments, the nucleic acid includes intemucleotide linkage modifications such as phosphate analogs having achiral and uncharged intersubunit linkages (e.g., Sterchak, E. P. et al., Organic Chem., 52:4202, (1987)), or uncharged morpholino-based polymers having achiral intersubunit linkages (see, e.g., U.S. Pat. No. 5,034,506). Some internucleotide linkage analogs include morpholidate, acetal, and polyamide-linked heterocycles. Other backbone and linkage modifications include, but are not limited to, phosphorothioates, peptide nucleic acids, tricyclo-DNA, decoy oligonucleotide, ribozymes, spiegelmers (containing L nucleic acids, an apatamer with high binding affinity), or CpG oligomers.

[0083] Phosphorothioates (or S-oligos) are a variant of normal DNA in which one of the nonbridging oxygens is replaced by a sulfur. The sulfurization of the internucleotide bond dramatically reduces the action of endo- and exonucleases including 5' to 3' and 3' to 5' DNA POL 1 exonuclease, nucleases SI and Pl, RNases, serum nucleases and snake venom phosphodiesterase. In addition, the potential for crossing the lipid bilayer increases. Because of these important improvements, phosphorothioates have found increasing application in cell regulation. Phosphorothioates are made by two principal routes: by the action of a solution of elemental sulfur in carbon disulfide on a hydrogen phosphonate, or by the more recent method of sulfurizing phosphite triesters with either tetraethylthiuram disulfide (TETD) or 3H-1, 2- bensodithiol-3-one 1, 1-dioxide (BDTD).4 The latter methods avoid the problem of elemental sulfur's insolubility in most organic solvents and the toxicity of carbon disulfide. The TETD and BDTD methods also yield higher purity phosphorothioates.

[0084] Peptide nucleic acids (PNA) are molecules in which the phosphate backbone of oligonucleotides is replaced in its entirety by repeating N-(2-aminoethyl)-glycine units and phosphodiester bonds are replaced by peptide bonds. The various heterocyclic bases are linked to the backbone by methylene carbonyl bonds. PNAs maintain spacing of heterocyclic bases that is similar to oligonucleotides but are achiral and neutrally charged molecules. Peptide nucleic acids are typically comprised of peptide nucleic acid monomers. The heterocyclic bases can be any of the standard bases (uracil, thymine, cytosine, adenine and guanine) or any of the modified heterocyclic bases described below. A PNA can also have one or more peptide or amino acid variations and modifications. Thus, the backbone constituents of PNAs may be peptide linkages, or alternatively, they may be non-peptide linkages. Examples include acetyl caps, amino spacers such as 8-amino-3,6-dioxaoctanoic acid (referred to herein as O-linkers), and the like. Methods for the chemical assembly of PNAs are well known.

[0085] In some embodiments, the nucleic acid includes one or more chemically-modified heterocyclic bases including, but are not limited to, inosine, 5-(l-propynyl) uracil (pU), 5-(l- propynyl) cytosine (pC), 5-methylcytosine, 8-oxo-adenine, pseudocytosine, pseudoisocytosine, 5 and 2-amino-5-(2'-deoxy-P-D-ribofuranosyl)pyridine (2-aminopyridine), and various pyrrolo- and pyrazolopyrimidine derivatives, 4-acetylcytosine, 8-hydroxy-N-6- methyladenosine, aziridinylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylamninomethyluracil, dihydrouracil, N6-isopentenyladenine, 1 -methyladenine, 1 -methylpseudouracil, 1 -methyl guanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3- methyleytosine, N6-methyladenine, 7-methyl guanine, 5-methylaminomethyluracil, 5- methoxy-aminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil- 5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5- oxyacetic acid methylester, 2,6-diaminopurine, and 2-modified analogs such as, but not limited to O-methyl, amino-, and fluoro-modified analogs. Inhibitory RNAs modified with 2'-fluoro (2'-F) pyrimidines appear to have favorable properties in vitro. Moreover, one report recently suggested 2'-F modified siRNAs have enhanced activity in cell culture as compared to 2'-OH containing siRNAs. 2'-F modified siRNAs are functional in mice but that they do not necessarily have enhanced intracellular activity over 2'-OH siRNAs.

[0086] In some embodiments the nucleic acid includes one or more sugar moiety modifications, including, but are not limited to, 2'-O-aminoethoxy, 2'-O-amonioethyl (2'- OAE), 2'-O-methoxy, 2'-O-methyl, 2-guanidoethyl (2'-0GE), 2'-0,4'-C-methylene (LNA), 2'- O-(methoxyethyl) (2'-0ME) and 2'-0 — (N-(methyl)acetamido) (2'-0MA).

[0087] Methods of gene therapy typically rely on the introduction into the cell of a nucleic acid molecule that alters the genotype of the cell. Introduction of the nucleic acid molecule can correct, replace, or otherwise alters the endogenous gene via genetic recombination. Methods can include introduction of an entire replacement copy of a defective gene, a heterologous gene, or a small nucleic acid molecule such as an oligonucleotide. For example, corrective gene can be introduced into a nonspecific location within the host's genome. This approach typically requires delivery systems to introduce the replacement gene into the cell, such as genetically engineered viral vectors.

[0088] Methods to construct expression vectors containing genetic sequences and appropriate transcriptional and translational control elements are well known in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Expression vectors generally contain regulatory sequences necessary elements for the translation and / or transcription of the inserted coding sequence. For example, the coding sequence can be operably linked to a promoter and / or enhancer to help control the expression of the desired gene product. Promoters used in biotechnology are of different types according to the intended type of control of gene expression. They can be generally divided into constitutive promoters, tissue-specific or development-stage-specific promoters, inducible promoters, and synthetic promoters. Viral vectors include adenovirus, adeno-associated virus, herpes virus, vaccinia virus, polio virus, AIDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including these viruses with the HIV backbone. Also useful are any viral families which share the properties of these viruses which make them suitable for use as vectors. Typically, viral vectors contain, nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome. When engineered as vectors, viruses typically have one or more of the early genes removed and a gene or gene / promoter cassette is inserted into the viral genome in place of the removed viral DNA.

[0089] Gene targeting via target recombination, such as homologous recombination (HR), is another strategy for gene correction. Gene correction at a target locus can be mediated by donor DNA fragments homologous to the target gene (Hu, et al., Mol. Biotech., 29: 197-210 (2005); Olsen, et al., J. Gene Med., 7: 1534-1544 (2005)). One method of targeted recombination includes the use of triplex -forming oligonucleotides (TFOs) which bind as third strands to homopurine / homopyrimidine sites in duplex DNA in a sequence-specific manner. Triplex forming oligonucleotides can interact with either double-stranded or single-stranded nucleic acids. When triplex molecules interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependent on both Watson- Crick and Hoogsteen base-pairing.

[0090] Methods for targeted gene therapy using triplex-forming oligonucleotides (TFO's) and peptide nucleic acids (PNAs) are described in U.S. Published Application No. 20070219122 and their use for treating infectious diseases such as HIV are described in U.S. Published Application No. 2008050920. The triplex-forming molecules can also be tail clamp peptide nucleic acids (tcPNAs), such as those described in U.S. Published Application No. 2011 / 0262406. Highly stable PNA:DNA:PNA triplex structures can be formed from strand invasion of a duplex DNA with two PNA strands. In this complex, the PNA / DNA / PNA triple helix portion and the PNA / DNA duplex portion both produce displacement of the pyrimidine- rich triple helix, creating an altered structure that has been shown to strongly provoke the nucleotide excision repair pathway and to activate the site for recombination with the donor oligonucleotide. Two PNA strands can also be linked together to form a bis-PNA molecule. The triplex -forming molecules are useful to induce site-specific homologous recombination in mammalian cells when used in combination with one or more donor oligonucleotides which provides the corrected sequence. Donor oligonucleotides can be tethered to triplex-forming molecules or can be separate from the triplex-forming molecules. The donor oligonucleotides can contain at least one nucleotide mutation, insertion or deletion relative to the target duplex DNA.

[0091] Double duplex-forming molecules, such as a pair of pseudocomplementary oligonucleotides, can also induce recombination with a donor oligonucleotide at a chromosomal site. Use of pseudocomplementary oligonucleotides in targeted gene therapy is described in U.S. Published Application No. 2011 / 0262406. Pseudocomplementary oligonucleotides are complementary oligonucleotides that contain one or more modifications such that they do not recognize or hybridize to each other, for example due to steric hindrance, but each can recognize and hybridize to complementary nucleic acid strands at the target site. In some embodiments, pseudocomplementary oligonucleotides are pseudocomplementary peptide nucleic acids (pcPNAs). Pseudocomplementary oligonucleotides can be more efficient and provide increased flexibility over methods of induced recombination such as triple-helix oligonucleotides and bis-peptide nucleic acids which require a polypurine sequence in the target double-stranded DNA.

[0092] Diagnostic Agents

[0093] Exemplary diagnostic materials include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides. Suitable diagnostic agents include, but are not limited to, x-ray imaging agents and contrast media. Radionuclides also can be used as imaging agents. Examples of other suitable contrast agents include gases or gas emitting compounds, which are radioopaque. PEG-BBs can further include agents useful for determining the location of administered particles. Agents useful for this purpose include fluorescent tags, radionuclides and contrast agents.

[0094] For those-embodiments where the one or more therapeutic, prophylactic, and / or diagnostic agents are loaded with and / or associated with the surface of the PEG-BB nanocarrier, the percent drug loading is from about 1% to about 80%, from about 1% to about 50%, including from about 1% to about 40% by weight, including from about 1% to about 20% by weight, including from about 1% to about 10% by weight. The ranges above are inclusive of all values from 1% to 80%.

[0095] Nutraceuticals

[0096] Nutraceuticals can also be incorporated. These may be vitamins, supplements such as calcium or biotin, or natural ingredients such as plant extracts or phytohormones.

[0097] Pharmaceutical Compositions

[0098] The formulations described herein contain an effective amount of PEG-BB nanocarrier in a pharmaceutical carrier appropriate for administration to a mucosal surface. The formulations can be administered parenterally (e.g., by injection or infusion), topically (e.g., to the eye), or via pulmonary administration.

[0099] Pulmonary Formulations

[0100] Pharmaceutical formulations and methods for the pulmonary administration of active agents to patients are known in the art.

[0101] The respiratory tract is the structure involved in the exchange of gases between the atmosphere and the blood stream. The respiratory tract encompasses the upper airways, including the oropharynx and larynx, followed by the lower airways, which include the trachea followed by bifurcations into the bronchi and bronchi oli. The upper and lower airways are called the conducting airways. The terminal bronchioli then divide into respiratory bronchioli which then lead to the ultimate respiratory zone, the alveoli, or deep lung, where the exchange of gases occurs.

[0102] Formulations can be divided into dry powder formulations and liquid formulations. Both dry powder and liquid formulations can be used to form aerosol formulations. The term aerosol as used herein refers to any preparation of a fine mist of particles, which can be in solution or a suspension, whether or not it is produced using a propellant.

[0103] Dry Powder Formulations

[0104] Dry powder formulations are finely divided solid formulations which are suitable for pulmonary administration. Such dry powder formulations can be administered via pulmonary inhalation to a patient without the benefit of any carrier, other than air or a suitable propellant, or a carrier can be present.

[0105] The carrier may include a bulking agent or a lipid or surfactant, such as natural surfactants such as dipalmitoylphosphatidylcholine (DPPC). Synthetic and animal derived pulmonary surfactants include:

[0106] Synthetic Pulmonary Surfactants

[0107] Exosurf — a mixture of DPPC with hexadecanol and tyloxapol added as spreading agents Pumactant (Artificial Lung Expanding Compound or ALEC) — a mixture of DPPC and PG KL-4 — composed of DPPC, palmitoyl-oleoyl phosphatidylglycerol, and palmitic acid, combined with a 21 amino acid synthetic peptide that mimics the structural characteristics of SP-B.

[0108] Venticute — DPPC, PG, palmitic acid and recombinant SP-C

[0109] Animal Derived Surfactants

[0110] Alveofact — extracted from cow lung lavage fluid

[0111] Curosurf — extracted from material derived from minced pig lung Infasurf — extracted from calf lung lavage fluid

[0112] Survanta — extracted from minced cow lung with additional DPPC, palmitic acid and tripalmitin

[0113] Exosurf, Curosurf, Infasurf, and Survanta are the surfactants currently FDA approved for use in the U.S.

[0114] The carrier may also include one or more stabilizing agents or dispersing agents. The pharmaceutical carrier may also include one or more pH adjusters or buffers. Suitable buffers include organic salts prepared from organic acids and bases, such as sodium citrate or sodium ascorbate. The pharmaceutical carrier may also include one or more salts, such as sodium chloride or potassium chloride.

[0115] Dry powder formulations can be prepared by blending one or more PEG-BB with one or more acceptable carriers. Optionally, additional active agents may be incorporated into the mixture as discussed below. The mixture is then formed into particles suitable for pulmonary administration using techniques known in the art, such as lyophilization, spray drying, agglomeration, spray coating, coacervation, low temperature casting, milling (e.g., airattrition milling (jet milling), ball milling), high pressure homogenization, and / or supercritical fluid crystallization.

[0116] An appropriate method of particle formation can be selected based on the desired particle size, particle size distribution, and particle morphology desired for the formulation. In some cases, the method of particle formation is selected so as to produce a population of particles with the desired particle size, particle size distribution for pulmonary administration. Alternatively, the method of particle formation can produce a population of particles from which a population of particles with the desired particle size, particle size distribution for pulmonary administration is isolated, for example by sieving.

[0117] It is known in the art that particle morphology affects the depth of penetration of a particle into the lung. Accordingly, dry powder formulations are processed into particles having the appropriate mass median aerodynamic diameter (MMAD), tap density, and surface roughness to achieve delivery of the one or more active agents to the desired region(s) of the lung.

[0118] Dry powder formulations can be administered as dry powder using suitable methods known in the art. Alternatively, the dry powder formulations can be suspended in the liquid formulation s described below and administered to the lung using methods known in the art for the delivery of liquid formulations.

[0119] Liquid Formulations Liquid formulations contain one or more PEG-BBs suspended in a liquid carrier.

[0120] Suitable liquid carriers include, but are not limited to distilled water, de-ionized water, pure or ultrapure water, saline, and other physiologically acceptable aqueous solutions containing salts and / or buffers, such as phosphate buffered saline (PBS), Ringer's solution, and isotonic sodium chloride, or any other aqueous solution acceptable for administration to an animal or human.

[0121] Liquid formulations can be isotonic relative to physiological fluids and of approximately the same pH, ranging e.g., from about pH 4.0 to about pH 7.4, including from about pH 6.0 to pH 7.0. The liquid carrier can include one or more physiologically compatible buffers, such as a phosphate buffers. One skilled in the art can readily determine a suitable saline content and pH for an aqueous solution for pulmonary administration.

[0122] Liquid formulations may include one or more suspending agents, such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone, gum tragacanth, or lecithin. Liquid formulations may also include one or more preservatives, such as ethyl or n-propyl p- hy droxyb enzoate .

[0123] In some cases, the liquid formulation may contain one or more solvents that are low toxicity organic (i.e. nonaqueous) class 3 residual solvents, such as ethanol, acetone, ethyl acetate, tetrahydrofuran, ethyl ether, and propanol. These solvents can be selected based on their ability to readily aerosolize the formulation. Any such solvent included in the liquid formulation should not detrimentally react with the one or more active agents present in the liquid formulation. The solvent should be sufficiently volatile to enable formation of an aerosol of the solution or suspension. Additional solvents or aerosolizing agents, such as a freon, alcohol, glycol, polyglycol, or fatty acid, can also be included in the liquid formulation as desired to increase the volatility and / or alter the aerosolizing behavior of the solution or suspension.

[0124] Liquid formulations may also contain minor amounts of polymers, surfactants, or other excipients well known to those of the art. In this context, “minor amounts” means no excipients are present that might adversely affect uptake of the one or more active agents in the lungs.

[0125] Aerosol Formulations

[0126] The dry powder and liquid formulations described above can be used to form aerosol formulations for pulmonary administration. Aerosols for the delivery of therapeutic agents to the respiratory tract are known in the art. The term aerosol as used herein refers to any preparation of a fine mist of solid or liquid particles suspended in a gas. In some cases, the gas may be a propellant; however, this is not required. Aerosols may be produced using a number of standard techniques, including as ultrasonication or high-pressure treatment.

[0127] In some embodiments, a dry powder or liquid formulation as described above is formulated into aerosol formulations using one or more propellants. Suitable propellants include air, hydrocarbons, such as pentane, isopentane, butane, isobutane, propane and ethane, carbon dioxide, chlorofluorocarbons, fluorocarbons, and combinations thereof. Suitable fluorocarbons include 1-6 hydrogen containing fluorocarbons, such as CHF2CHF2, CF3CH2F, CH2F2CH3, and CF3CHFCF3 as well as fluorinated ethers such as CF3 — O — CF3, CF2H — O — CHF2, and CF3 — CF2 — O — CF2 — CH3. Suitable fluorocarbons also include perfluorocarbons, such as 1-4 carbon perfluorocarbons including CF3CF3, CF3CF2CF3, and CF3CF2CF2CF3.

[0128] In some embodiments, the propellants include, but not limited to, one or more hydrofluoroalkanes (HFA). Suitable HFA propellants, include but are not limited to, 1,1,1,2,3,3,-heptafluoro-n-propane (HFA227), 1,1,1,2-tetrafhroroethane (HFA 134) 1,1, 1,2, 25 3,3,3-heptafluoropropane (Propellant 227), or any mixture of these propellants.

[0129] In some embodiments, the one or more propellants have sufficient vapor pressure to render them effective as propellants. In some embodiments, the one or more propellants are selected so that the density of the mixture is matched to the density of the particles in the aerosol formulation in order to minimize settling or creaming of the particles in the aerosol formulation. The propellant can be present in an amount sufficient to propel a plurality of the selected doses of the aerosol formulation from an aerosol canister.

[0130] Devices for Pulmonary Administration

[0131] In some cases, a device is used to administer the formulations to the lungs. Suitable devices include, but are not limited to, dry powder inhalers, pressurized metered dose inhalers, nebulizers, and electrohydrodynamic aerosol devices.

[0132] Inhalation can occur through the nose and / or the mouth of the patient. Administration can occur by self-administration of the formulation while inhaling or by administration of the formulation via a respirator to a patient on a respirator.

[0133] Parenteral Formulations

[0134] In some embodiments, the PEG-BB are formulated for parenteral delivery, such as injection or infusion, in the form of a solution or suspension. The formulation can be administered via any route, such as, the blood stream or directly to the organ or tissue to be treated.

[0135] “Parenteral administration”, as used herein, means administration by any method other than through the digestive tract or non-invasive topical or regional routes. For example, parenteral administration may include administration to a patient intravenously, intradermally, intraperitoneally, intrapleurally, intratracheally, intramuscularly, subcutaneously, subjunctivally, by injection, and by infusion.

[0136] Parenteral formulations can be prepared as aqueous compositions using techniques is known in the art. Typically, such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions, and microemulsions thereof, liposomes, or emulsomes.

[0137] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, com oil, sesame oil, etc.), and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and / or by the use of surfactants. In some embodiments, isotonic agents, for example, sugars or sodium chloride, are included.

[0138] Solutions and dispersions of the active compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, and combination thereof.

[0139] Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Pol oxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-O-alanine, sodium N-lauryl-n- iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

[0140] The formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the active agent(s).

[0141] The formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.

[0142] Water soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.

[0143] Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles are well known in the art.

[0144] Topical Formulations

[0145] In still other embodiments, the PEG-BB are formulated for topical administration to mucosa. Suitable dosage forms for topical administration include creams, ointments, salves, sprays, gels, lotions, emulsions, liquids, and transdermal patches. The formulation may be formulated for transmucosal, transepithelial, transendothelial, or transdermal administration. The compositions contain one or more chemical penetration enhancers, membrane permeability agents, membrane transport agents, emollients, surfactants, stabilizers, and combination thereof. In some embodiments, the PEG-BBs can be administered as a liquid formulation, such as a solution or suspension, a semi-solid formulation, such as a lotion or ointment, or a solid formulation. In some embodiments, the PEG-BBs are formulated as liquids, including solutions and suspensions, such as eye drops or as a semi-solid formulation, such as ointment or lotion for topical application to mucosa, such as the eye or vaginally or rectally.

[0146] The formulation may contain one or more excipients, such as emollients, surfactants, emulsifiers, penetration enhancers, and the like.

[0147] Enteral Formulations

[0148] Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be made using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can prepared as hard or soft capsule shells, which can encapsulate liquid, solid, and semi-solid fill materials, using techniques well known in the art.

[0149] Formulations may be prepared using one or more pharmaceutically acceptable excipients, including diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.

[0150] Excipients, including plasticizers, pigments, colorants, stabilizing agents, and glidants, may also be used to form coated compositions for enteral administration. Delayed release dosage formulations may be prepared as described in standard references such as “Pharmaceutical dosage form tablets”, eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), “Remington — The science and practice of pharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, Md., 2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6th Edition, Ansel et al., (Media, Pa.: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.

[0151] The PEG-BBs can be coated, for example to delay release once the particles have passed through the acidic environment of the stomach. Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

[0152] Diluents, also referred to as “fillers,” are typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar.

[0153] Binders are used to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / polymethacrylic acid and polyvinylpyrrolidone.

[0154] Lubricants are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, tale, and mineral oil.

[0155] Disintegrants are used to facilitate dosage form disintegration or “breakup” after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross-linked polymers, such as crosslinked PVP (Polyplasdone® XL from GAF Chemical Corp).

[0156] Stabilizers are used to inhibit or retard drug decomposition reactions that include, by way of example, oxidative reactions. Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E, tocopherol and its salts; sulfites such as sodium metabisulphite; cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxyanisole (BHA).

[0157] The following non-limiting examples are provided to further illustrate the present invention.

[0158] EXAMPLES

[0159] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.

[0160] EXAMPLE I

[0161] Introduction It is highly desired for a delivery system that sneaks through not only the physical and biochemical barriers of mucus but also the tight periciliary brush gel to be efficiently internalized by epithelial cells.

[0162] Provided herein is a wormlike PEG-based polymeric nanocarrier that can rapidly translocate across all barriers within the human airway surface. A nanocarrier of a long linear backbone densely grafted by many (-1,000) low molecular weight (MW) PEG side chains (-1,000 g / mol) was designed and synthesized. This results in a bottlebrush PEG (PEG-BB) macromolecule featuring a contour length of -250 nm, a cross-section of -20 nm, and a hydrodynamic diameter of -40 nm. Using the classic human bronchial epithelial cell (HBEC) culture as a model system, it is shown that PEG-BB can rapidly penetrate through endogenous airway mucus and the periciliary brush layer to be internalized by epithelial cells across the whole epithelium. By quantifying the cellular uptake of polymeric carriers of various molecular architectures and manipulating cell proliferation and endocytosis pathways, it is shown that translocation of PEG-BB across the epithelium is driven by bottlebrush-architecture enhanced endocytosis. The results demonstrate that large, wormlike PEG-BB polymers can be used as a novel carrier for pulmonary and mucosal drug delivery.

[0163] Material and Methods

[0164] Materials for polymer synthesis.

[0165] Fluorescein o-acrylate (Flu, 95%), 2-methoxyethyl acrylate (MEA, 98%, monomethyl ether hydroquinone (MEHQ) as inhibitor), and polyethylene glycol) methyl ether methacrylate (MEMA-PEG, 950 g / mol, MEHQ as inhibitor) were purchased from Sigma Aldrich and purified by recrystallizing in acetone to remove inhibitors. 2- (dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT, 98%), 2,2 ’-azobi s(2- methylpropionitrile) (AIBN, 98%) and N, A-dimethylformamide (DMF, A99.8%) are purchased from Sigma Aldrich and used as received.

[0166] Synthesis of PEG-based carriers.

[0167] I. Densely grafted bottlebrush PEG (PEG-BB).

[0168] A 25 mL Schlenk flask was charged with MEMA-PEG (2.28g, Immol, 1200 eq), Flu (12eq), DDMAT (1 eq), AIBN (0.2 eq), and 6 mL DMF. The mixture was degassed using three freeze-evacuate-thaw cycles and then the flask was sealed under nitrogen. The sealed flask was immersed in a heated oil bath at 70 °C for 12 hours and then the reaction was stopped by exposing the solution to air, at which timepoint the conversion of the polymerization is 82.5%, as confirmed by 'H NMR FIG. S2). At this conversion, the DP of PEG is 990, and the DP of Flu is about 10. The reaction mixture was precipitated in ethyl ether three times to remove unreacted monomers and other impurities. Using a dialysis tube with a molecular-weight-cutoff (MWCO) of 3.5 kDa, PEG-BB was further purified through dialysis against water for 3 days. The solution was freeze-dried for 3 days to obtain the final product, which was a light-yellow powder.

[0169] II. Loosely grafted PEG (PEG-LG).

[0170] A 25 mL Schlenk flask was charged with MEMA-PEG (2.14g, 2.25mmol, 750eq), MEA (1.85g, 11.4mmol, 3800eq), Flu (45.5eq), DDMAT(leq), AIBN (0.2eq), and 10 mL DMF. The mixture was degassed using three freeze-evacuate-thaw cycles and then the flask was sealed under nitrogen. Then, the sealed flask was placed in a heated oil bath (70 °C) for 12 hours. The reaction was stopped by exposing the solution to air, with the final DP of PEG is about 750. The reaction mixture was precipitated in ethyl ether and dialysis against water for 3 days using tubes with a pore size molar mass cutoff of 3.5 kDa. Then, the solution was freeze dried under vacuum for 3 days to get the final product. The final molar ratio between MEMA-PEG and MEA is 1 :5, as confirmed byJH NMR (Fig. S3).

[0171] Characterization of PEG-based carriers.

[0172] I,!H NMR characterization.

[0173] Proton nuclear magnetic resonance (JH NMR) spectroscopy was performed using Varian NMRS 600 MHz spectrometer. For all samples, deuterated chloroform (CDCh) wss used as a solvent, except for PEG-LG which is analyzed using deuterated water (D2O).

[0174] II, Gel permeation chromatography (GPC) characterization.

[0175] GPC measurements were conducted using TOSOH EcoSEC HLC-8320 GPC system equipped with two TOSOH Bioscience TSKgel GMHHR-M 5 pm columns in series. The GPC system includes a refractive index detector and operates at a temperature of 40 °C. High- performance liquid chromatography (HPLC) grade trifluoroacetic acid (TFA) was used as the eluent, and it was delivered at a flow rate of 1 mL / min. The samples for analysis were prepared by dissolving them in TFA at a concentration of approximately 5 mg / mL.

[0176] III, Dynamic light scattering (PLS). DLS and (^-potential measurements were performed on a Malvern Zetasizer Ultra with a 4.0 mW laser (633 nm) at different temperatures. Samples were dissolved in water with a concentration of 0.2 mg / ml and filtered (0.45 pm, PTFE) before measurement. Size measurements were performed in square DTS0012 cuvettes (Malvern) in triplicate. For PEG-BB and PEG-LG, the (^-potential values are -8.8 mV and -2.0 mV, respectively.

[0177] Human bronchial epithelial cell (HBEC) culture. Primary HBECs were obtained from Marsico Lung Institute Tissue Procurement and Cell Culture Core at the University of North Carolina at Chapel Hill, under protocol number 194 03-1396 approved by the UNC Biomedical Institutional Review Board. For statistics, HBECs from 5 non-smoker (NS) donors without a history of chronic lung diseases (age / sex / race: 49 / Female / Caucasian, 17 / Male / Caucasian, 30 / Female / Hispanic,

[0178] 27 / Female / Caucasian, 22 / Female / Caucasian) were used. For cell expansion, HBECs were cultured using PneumaCult™-Ex Plus Medium (STEMCELL Technologies, Cat. No. 05040). HBECs were passaged using Accutase™ Cell Detachment Solution (Innovative Cell Technologies, Cat. No. AT 104). For all experiments, passage 2 cells were used, beyond which HBECs may lose their sternness27. HEBCs were seeded on Transwell® inserts (Corning, Cat. No. 3460) at the density of 4.2* 104cells / well and expansion medium (PneumaCult™-Ex Plus Medium, STEMCELL Technologies, Cat. No. 05040) was added to both the basal and apical chambers. After 5-7 days, HBECs reach over 90% confluence. The cultures were transferred to ALI by removing the apical medium and replacing the expansion medium with ALI culture medium (PneumaCult™-ALI Medium, STEMCELL Technologies, Cat. No. 05001). The medium was changed every other day. After 2 weeks of ALI culture, mucus starts to accumulate and was washed three times per week. To wash off mucus, 500 pL of Dulbecco’s phosphate- buffered saline (DPBS, Gibco, Cat. No. 14-200-075) was added to each insert, the cell culture was incubated for 15 min, and the DPBS was aspirated in the apical chamber. The washing process was repeated three times. After 4 weeks of ALI culture, HBECs were fully differentiated. Based on a previous study5,6, the mucus was allowed to accumulate for approximately 2 weeks without washing, reaching a concentration of -14% solids and a height of -15 pm.

[0179] Measurement of the translocation of PEG-based carriers across the airway epithelial layer.

[0180] To measure the uptake of PEG-BB from the apical side, 10 pl of 1 mg / ml PEG-BB was added to the apical side of each HBEC culture, the culture was incubated for about 1 min, and then the culture was rinsed with pre-warmed DPBS to wash off any remaining PEG-BB. Fluorescence confocal microscopy (Leica, SP8) was used to quantify the uptake of fluorescent PEG-BB. The confocal microscope was equipped with an environmental chamber, which has a controlled temperature at 37°C, CO2 at 5%, and humidified air at 2L / hr, to allow for longtime live-cell imaging. For fluorescein, a 512 nm laser was used for excitation, and a bandwidth of 500-600 nm was used for emission. Using Z-stack scanning, the full thickness of the cell body of the airway epithelium was imaged at the step size of 1.12 pm with a total of 15 frames. The periciliary layer was about 7 pm5. Therefore, the whole airway epithelium was around 23 pm thick, consistent with the literature value28.

[0181] To explore the cellular uptake over longer durations, 10 pl of 1 mg / ml PEG-BB was added to the apical side and HBECs were incubated overnight. Imaging was performed to show the distribution of PEG-BB molecules without washing off mucus. After performing initial confocal microscopy with intact mucus, 10 pl of 100 pg / ml 70 kDa Texas Red™ dextran (Thermo Fisher Scientific, Cat. No. D1830) solution was added to the apical side for about 1~2 hours. The 70 kDa dextran molecules penetrated the periciliary layer but cannot cross the epithelial layer, allowing one to delineate the boundary of the epithelial surface5. Confocal microscopy was used to image the full profile of the culture using XZ scanning mode and to image the fluorescence of the cells at the apical focal plane using XY scanning mode.

[0182] To explore the uptake of PEG-BB from the basal side of airway epithelium, we add PEG-BB molecules to the culture medium in the basal chamber to reach a concentration of 100 pg / ml, incubate the HBEC culture overnight, and change the medium on the next day to remove any free PEG-BB. We use confocal microscopy to capture fluorescence at the apical focal plane.

[0183] To determine the effect of molecular architecture on the uptake of PEG, the absorption of loosely grafted PEG (PEG-LG) by HBECs from the apical side was studied. 10 pl of 1 mg / ml PEG-LG was added to the apical side of HBECs with overnight incubation. Imaging was performed to show the distribution of PEG-LG molecules without washing off mucus. A similar procedure was used to label the periciliary layer followed by confocal imaging.

[0184] Imaging of PEG-BB in HBEC cytoplasm.

[0185] Differentiated airway epithelial cells were detached from the Transwell membrane by incubating HBECs with 1 ml of Accutase™ Cell Detachment Solution per well for 15 min at 37 °C. 200 pl of PneumaCult™-ALI Medium was added to a rectangle coverslip and a 1000 pl pipette tip was used to scrape a full thickness of airway epithelium into the medium. Another rectangle coverslip was used to cover the medium containing the airway epithelial sample. Afterward, the sample was mounted onto a 63 X oil objective (NA 1.4) with pre-applied lens oil for confocal microscopy. For fluorescein, a 512 nm laser was used for excitation and a bandwidth of 500-600 nm was used for emission.

[0186] NIH-3T3 cell culture, imaging of PEG-BB fluorescence, and mitosis inhibition by colchicine. For the culture of NIH-3T3 cells, Dulbecco’s Modified Eagle Medium (Corning, Cat. No. 10-013-CV) was supplemented with 10% Fetal Bovine Serum (Life Technologies Corporation, Cat. No. A3160401). The medium was changed every other day. Cells were passaged using Accutase® Cell Detachment Solution (Innovative Cell Technologies, AT 104).

[0187] To study the internalization of PEG-BB by NIH-3T3 fibroblasts, PEG-BB was added to the culture medium to reach a concentration of 100 pg / ml and cells were incubated overnight. On the next day, the culture medium was replaced with fresh medium to wash off any free PEG-BB. After washing, cell nuclei were stained by adding Hoechst 33342 (Thermo Fischer Scientific, 62249) to medium at the concentration of 20 pg / ml and incubating NIH-3T3 fibroblasts for 5 min at 37 °C, followed by rinsing the cells twice with DPBS to wash away remaining Hoechst 33342 molecules. Confocal microscopy was performed to image intracellular PEG-BB fluorescence.

[0188] To inhibit mitosis without impairing the viability of NIH-3T3 fibroblasts, 20 ng / ml colchicine (Thermo Fisher Scientific, Cat. No. 227120010) was used to treat NIH-3T3 fibroblasts for 30 minutes at 37 °C before treatment with PEG-BB29. The same protocols as above to incubate NIH-3T3 fibroblast with PEG-BB, stain nuclei, and image intracellular PEG- BB fluorescence was used.

[0189] For each NIH-3T3 culture, a 10 X dry objective was used to image a region of interest (ROI) with a dimension of 381.5x381.5 pm2. The sequential scanning mode was used to avoid overlap of fluorescence. For fluorescein, a 512 nm laser was used for excitation and a bandwidth of 500-600 nm was used for emission. For Hoechst 33342, a UV laser of 405 nm was used for excitation and a bandwidth of 400-500 nm was used for emission.

[0190] Measurement of the cell number, fluorescent cell fraction, fluorescence index, and fluorescent area fraction.

[0191] The cell number was counted based on the number of nuclei stained by Hoechst 33342. Relative cell count (G-), defined as the ratio of cell counts normalized to the initial cell number on Day 1, to present the cell number on each day was used.

[0192] Fluorescent cell fraction (Fc) was defined as the ratio of fluorescent cells to the total cell count. Regardless of the size or intensity of fluorescence, cells with any fluorescence were counted. For each condition, 5 parallel wells of cell culture were used for statistical analysis. Fluorescence index (P) is defined as P = FcX Cr. Fluorescent area fraction (Fa) was derived by the fluorescent area (Af) divided by the cytoplasm area. The cytoplasm area was the cell area Ac) minus the nuclear area (An). For each condition and timepoint, 100 cells were randomly chosen for statistical analysis.

[0193] Uptake of PEG-BB, 2 MDa dextran, 70 kDa dextran, and PEG-LG by NIH-3T3 fibroblast cells.

[0194] PEG-BB, 2 MDa FITC dextran (Millipore Sigma, Cat. No. FD2000S), 70 kDa FITC dextran (Millipore Sigma, Cat. No. FD70), and PEG-LG were added to the culture medium at the same concentration of 100 pg / ml to incubate with NIH-3T3 cells overnight respectively. After incubation, 20 pg / ml Hoechst 33342 was used to incubate NIH-3T3 cells for 5 min at 37 °C to stain the cell nuclei and then DPBS was used to rinse the culture twice. After rinsing, the same amount of 2 MDa FITC dextran, 70 kDa FITC dextran, and PEG-LG were re-added for pre-washing imaging using confocal microscopy. Washing with pre-warmed medium was performed twice, followed by post-washing imaging using confocal microscopy.

[0195] Effects of endocytosis inhibitors on the uptake of PEG-BB by NIH-3T3 fibroblasts and HBECs.

[0196] 5 pg / ml chlorpromazine (Thermo Fisher Scientific, Cat. No. J63659), 5 pg / ml nystatin (Thermo Fisher Scientific, Cat. No. BP29495), and 0.1 pg / ml wortmannin (Thermo Fisher Scientific, Cat. No. W0007) was added to the culture medium to incubate NIH-3T3 fibroblasts and HBECs for 4 hours at 37 °C, to inhibit endocytosis. After inhibiting endocytosis, the cellular uptake of PEG-BB by NIH-3T3 fibroblasts and HBECs was studied using the same protocols described above.

[0197] To study how inhibition of endocytosis affects PEG-BB uptake by NIH-3T3 cells, 100 mg / ml PEG-BB was added to the culture medium, and cells were incubated overnight. On the next day, cell nuclei were stained by adding Hoechst 33342 to the culture medium at a concentration of 20 pg / ml and NIH-3T3 cells were incubated for 5 min at 37 °C, followed by rinsing the cells twice with DPBS to wash away free Hoechst 33342. Confocal microscopy was performed to capture both extracellular and intracellular PEG-BB fluorescence if any. After initial imaging with the presence of extracellular PEG-BB in the culture medium, fresh medium was used to wash off any free PEG-BB. After washing, confocal microscopy was performed again to image intracellular PEG-BB fluorescence if any.

[0198] To study the uptake of PEG-BB by HBECs from the apical and basal sides, 10 pl of 1 mg / ml PEG-BB was added to the apical side and 100 pg / ml PEG-BB was added to the basal medium respectively. After overnight incubation, the cell culture was not washed to keep the mucus layer intact, and confocal microscopy was used to capture fluorescence at the apical focal plane for PEG-BB added from both apical and basal sides.

[0199] Statistical analysis.

[0200] Statistical analysis was performed using one-way analysis of variance (ANOVA). For the post hoc test after performing statistical analysis, Tukey’s honestly significant difference (HSD) test was used to determine the significant differences between groups. p>0.05 was considered statistically significant.

[0201] Results

[0202] Design and synthesis of PEG-based nanocarriers with different molecular architectures

[0203] The design of the nanocarrier is inspired by the molecular structure of mucins, featuring a large polypeptide backbone that is heavily glycosylated with many sugar chains30 32. A mucin-like PEG-based polymer was designed, which comprises of a long linear backbone densely grafted by many relatively short PEG side chains. Because the side chains highly overlap with each other, the only way for them to avoid molecular crowding is to extend radially away from the backbone, forming a bottlebrush architecture as illustrated in FIG. 1C and FIG. SI. Analogous to “sausage versus spaghetti”, the bottlebrush polymer is essentially a wormlike ‘fat’ linear polymer33 37. It was hypothesized that the grafting density of PEG side chains can be precisely controlled to enable a non-sticky carrier, whereas the flexibility and wormlike geometry of the bottlebrush carrier allow it to sneak through the tight mesh of mucus and periciliary gels to be internalized by epithelial cells.

[0204] To test the hypothesis, a PEG-BB nanocarrier was designed with a precisely controlled molecular architecture, denoted by three parameters, [nsc, s, ], where nscis the number of PEG side chains, s is the molar ratio of spacer monomers to the side chains, and f is the fraction of fluorescent probes (FIG. 1C). The side chains are low MW methacrylate-terminated PEG of 950 g / mol; this value is the same as that used for mucus-penetrating PEGylated nanoparticles38. 2-methoxyethyl acrylate (MEA) was used as the spacer monomer, as MEA is chemically similar to PEG but has a much lower MW of 130 g / mol, so that it does not alter the chemical nature of the PEG carrier and only reduces the grafting density of PEG side chains. Fluorescein O-methacrylate was used as the fluorescent probe and fixed its fraction at 1% to ensure relatively bright fluorescence.

[0205] Using reversible addition fragmentation chain transfer radical polymerization (RAFT)39, a living polymerization technique widely used for controlled polymer synthesis, the side chain, spacer monomer, and fluorescent probe were copolymerized at prescribed ratios to create fluorescent PEG-based nanocarriers (FIG. 1C). The PEG-BB consists of 990 side chains but no spacers ([990, 0, 0.01]), as illustrated by the upper panel of FIG. ID. Successful synthesis is confirmed by proton nuclear magnetic resonance ( 'H-NMR) spectroscopy (FIG. S2) and gel permeation chromatography (GPC) (FIG. IE). The linear grafting density of PEG side chains is very high, with four side chains per nanometer (4 nm'1), so that PEG-BB is wormlike with an effective monomer size about the bottlebrush cross-section, be— 20 nm (theory in Example II, FIG. SI). In a good solvent such as water, the conformation of PEG- BB adopts a self-avoiding random walk of constituent effective monomers. Thus, the radius of gyration of PEG-BB is Rg« « 36 nm, where a « 0.4 is a prefactor that relates Rgto the end-to-end distance Ro« beLmax / be)3, / 5of a linear polymer in good solvent40, and Lmax« nscl « 250 nm is the contour length with I = 0.254 nm being the length of a repeating unit along the bottlebrush backbone. This theoretical value is consistent with the hydrodynamic diameter of PEG-BB, dh= 37 nm, measured by dynamic light scattering (DLS) (solid line in FIG. IF; Example II). Further, it was verified that the size of PEG-BB is nearly independent of temperature within the range between 20 °C and 45 °C (FIG. 1G). Because the PEG-BB has a hydrodynamic diameter that is large enough to be excluded from the periciliary brush yet has a cross-section smaller than the average mesh size of the periciliary brush gel, this polymer allows one to test whether the wormlike geometry allows the translocation of the PEG-BB across the human airway surface barriers.

[0206] Bottlebrush architecture enables rapid translocation of PEG-based nanocarriers across human airway surface barriers

[0207] The classic air-liquid-interface (ALI) culture system was used to model human airway surface barriers41. In the ALI system, HBECs are cultured on a porous plastic membrane, through which nutrients are transported from the cell culture medium on the basal side, whereas on the apical side, cells are in contact with air27. After approximately 4 weeks, primary HBECs, or human airway basal cells, differentiate into ciliated cells and goblet cells, forming a pseudostratified columnar epithelium that recapitulates essential biological features of the human airway epithelium28 42 44. Specifically, the pseudostratified airway epithelium consists of three layers: (i) an intact endogenous mucus hydrogel layer, (ii) a periciliary layer that separates the mucus hydrogel from the epithelial cells, and (iii) a layer of epithelial cells connected by cell junctions (FIG. 2A)45.

[0208] The uptake of PEG-BB molecules by HBECs from the apical side, where both the mucus hydrogel and the periciliary brush are present to serve as barriers for the delivery of drugs via inhalation46, was explored first. Based on a previous study5,6, for well-differentiated HBEC cultures, the mucus was allowed to accumulate for two weeks, at which timepoint the mucus reaches a concentration of -14% (solids) and a height of -15 pm; this corresponds to approximately 1.5 pl mucus per well. To each well, 10 pl of 1 mg / ml PEG-BB was added from the apical side so that the final mucus concentration is approximately 2% (solids), comparable to that of healthy mucus. The culture was incubated for about 1 minute and any remaining PEG-BB was washed off using pre-warmed Dulbecco’s phosphate-buffered saline (DPBS), as illustrated by FIG. 2A. Using fluorescence confocal microscopy, the profile of PEG-BB was then immediately imaged across the whole epithelial layer. Within such a short period of incubation, HBECs at the apical focal plane exhibit pronounced fluorescence (FIGS. 2B, 21), indicating rapid uptake of PEG-BB by HBECs. These results suggest that PEG-BB molecules can easily penetrate through the mucus and periciliary gels to be internalized by HBECs.

[0209] Interestingly, for the cells that contain PEG-BB molecules, the distribution of PEG-BB molecules within individual cells dramatically changes with the depth within the epithelial layer. At the apical focal plane, PEG-BB molecules spread the cross-area of the whole cell, as reflected by the nearly homogenous fluorescence bounded within the contour of the cell crosssection (dashed line, FIGS. 2B, 21). However, as the focal plane moves from the apical to the basal side, the fluorescence area within individual cells dramatically decreases, as shown in FIG. 2B. This is likely because the incubation time -1 min is too short for PEG-BB molecules to reach the basal side of the epithelial layer.

[0210] To explore the cellular uptake over longer durations, after adding PEG-BB to the apical side, HBECs were incubated overnight and then the distribution of PEG-BB molecules was imaged without washing off mucus (FIG. 2C). It was found that the fluorescence of PEG-BB is heterogeneously present within cells on the apical focal plane, as shown in FIG. 2D. To further explore the distribution of PEG-BB across the epithelial layer, 70 kDa Texas Red™ dextran was added to the apical side. As established in a previous study5, these molecules penetrate the periciliary layer but cannot cross the epithelial layer, allowing one to delineate the boundary of the epithelial surface, as confirmed by a red fluorescence layer in the middle panel of FIG. 2E. Within the periciliary layer, cilia exhibit green fluorescence, suggesting that PEG-BB molecules can accumulate in the periciliary layer and fluorescently label cilia (FIG. 2E). Within the epithelial layer, PEG-BB molecules are nearly homogenously distributed, as shown by the bright green fluorescence across the whole epithelium (FIG. 2F). By dissociating the pseudostratified epithelium from the Transwell plastic membrane to individual cells, it was confirmed that PEG-BB molecules are internalized by HBECs; however, PEG-BB molecules are not present in the nucleus and only present in the cytoplasm (FIG. 2G). Further, the side view of the epithelial layer reveals that PEG-BB is within epithelial cells from the apical to the basal side. These results demonstrate the cellular uptake of PEG-BB molecules and their ability to penetrate through the whole epithelial layer from the apical side.

[0211] Next, the uptake of PEG-BB was explored from the basal side of the airway epithelium, a process needed for the uptake of intravenously administered drugs. PEG-BB molecules were added to the culture medium on the basal side to reach a concentration of 100 pg / ml, the HBEC culture was incubate overnight, and the medium was changed to remove any free PEG-BB (FIG. 3A). It was found that the cells at the apical focal plane exhibit bright fluorescence (FIG. 3B). Moreover, PEG-BB molecules are present within the whole epithelial layer (FIG. 3C). Interestingly, compared to the uptake from the apical side, for the uptake from the basal side, although PEG-BB molecules are less abundant within the epithelial cells, they accumulate within the periciliary layer (FIG. 3C). This accumulation is further supported by the observation that cilia are also visible due to the presence of PEG-BB (FIG. 3D). Nevertheless, these results demonstrate the uptake of PEG-BB by HBECs from the basal side to the apical side.

[0212] To determine whether the uptake of PEG-BB molecules is because of their unique bottlebrush molecular architecture, a loosely grafted PEG polymer (PEG-LG) was synthesized with two neighboring PEG macromonomers separated by five spacer monomers on average ([750, 5, 0.01]), as illustrated by the lower panel of FIG. ID and confirmed by1H-NMR (FIG. S3) and GPC (dashed line, FIG. IF). The number of side chains (nsc= 750) is less than that of PEG-BB (nsc= 990) to compensate for the contribution of MW by the spacer. Despite that the contour length of PEG-LG (-1000 nm) is nearly four times of PEG-BB, the grafting density is relatively low of 0.65 nm’1, so that the side chains are far apart enough not to experience molecular crowding. As a result, the conformation of PEG-LG is coil-like with a hydrodynamic diameter of 31 nm, comparable to that of PEG-BB (FIG. IE). Similar to PEG-BB, the size of PEG-LG is nearly independent of temperature (FIG. IF). Following the same protocol for studying cellular uptake of PEG-BB, the uptake of PEG-LG by HBECs was quantified from the apical side and it was found that PEG-LG fluorescence on the apical focal plane is notably reduced (FIG. 3E). Profiling the penetration of PEG-LG across the whole epithelial layer further confirms minimum uptake of PEG-LG molecules, as shown by the negligible green fluorescence within the epithelial cells in FIG. 3F.

[0213] To quantitatively compare the uptake of PEG-BB and PEG-LG by HBECs, fluorescence fraction was introduced, a parameter that is defined as the PEG fluorescence area divided by the total cell area, which is equal to the image area as the cells are confluent. For the immediate uptake of PEG-BB within ~1 min from the apical side, the fluorescence fraction decreases from 48 ± 11% to 6 ±2% as the focal plane moves from the apical to the basal side (FIG. 3G). For overnight uptake, no matter if PEG-BB molecules are added from the apical side or from the basal side, there is no significant difference in fluorescence fraction at the apical focal plane (FIG. 3G). However, changing the molecular architecture from PEG-BB to PEG-LG results in a significant reduction of fluorescence fraction from 32 ± 5% to 3 ± 1% (FIG. 3G). These results demonstrate that the bottlebrush architecture significantly enhances the uptake of PEG-based carriers by HBECs.

[0214] Retention of PEG-BB molecules within cells

[0215] To explore the retention of PEG-BB molecules within cells, the cellular uptake of PEG- BB by single NIH-3T3 fibroblasts, a widely used cell line that allows for easy fluorescence staining and manipulation of biological pathways, was quantified. PEG-BB was added into the culture medium to reach a concentration of 100 pg / ml, NIH-3T3 cells were incubated overnight, and the medium was replaced to wash off free PEG-BB molecules if any. Simultaneously, Hoechst 33342 was used to stain the genomic DNA, or nuclei, of the cells. On Day 1, it was found that all NIH-3T3 fibroblasts are fluorescent regardless of variations in the area and intensity of fluorescence among individual cells (FIG. 4A). However, the number of fluorescent NIH-3T3 fibroblasts dramatically decreases as the culture time increases from Day 1 to Day 3, as visualized by the fluorescence images in FIG. 4A. To quantify the number of fluorescent cells, fluorescent cell fraction Fc, a parameter that is defined as the ratio of the number of fluorescent cells to the total cell count, was introduced. Specifically, cells with any fluorescence regardless of the fluorescence area or intensity are counted. As the culture time increases from Day 1 to 3, the value of Fcdecreases nearly linearly from 100% to 51 ±4%, as shown in FIG. 4C. These results show the uptake of PEG-BB across the cell membrane, but that the retention of PEG-BB within the cells decays with time.

[0216] To determine the role of cell proliferation in the reduction of PEG-BB fluorescence, colchicine was used to inhibit cell proliferation. Because colchicine inhibits mitosis by disrupting tubulin polymerization29, colchicine is cytotoxic and can be lethal to cells at a high dose47. To maintain cell viability while inhibiting mitosis, a colchicine concentration of 20 ng / ml29was used to treat NIH-3T3 fibroblasts before adding PEG-BB. Relative cell count was used, Cr, the cell count normalized to that of Day 1, to quantify the proliferation of NIH-3T3 fibroblasts. Without the treatment of colchicine, Cr doubles on Day 2 and triples on Day 3, as shown by the green squares in FIG. 4D. By contrast, with the treatment of colchicine, Cr remains constant at 1 across Days 1, 2, and 3, as shown by FIG. 4D. These results validate that the concentration of 20 ng / ml colchicine is adequate to inhibit mitosis without compromising cell viability. With cell proliferation inhibited by colchicine, all NIH-3T3 fibroblasts retain PEG-BB fluorescence across three days without a noticeable decrease in both fluorescence intensity and area among individual cells, as shown by the fluorescence images in FIG. 4B and circles in FIG. 4C. These results indicate that the reduction of intracellular PEG-BB is due to cell proliferation. This understanding is further supported by DAPI fluorescence of cell nuclei, which decreases progressively without colchicine treatment (FIG. 4A) but remains nearly constant after inhibiting cell proliferation (FIG. 4B).

[0217] To further explore the effects of cell proliferation on the retention of intracellular PEG- BB, fluorescence index was introduced, which is the product of fluorescent cell fraction and relative cell count: P=Fc'*Cr. This parameter describes the total number of cells with intracellular PEG-BB. As expected, after cell proliferation is inhibited by colchicine, the value of P remains constant across three days, as shown by the circles in FIG. 4E. By contrast, for the cells that proliferate, the value of P increases by nearly 1.5 times from Day 1 to Day 2; this suggests that PEG-BB molecules are passed to daughter cells during proliferation. Interestingly, at a longer incubation time on Day 3, despite an apparent decrease in fluorescent intensity within individual cells (images within the lower two rows in FIG. 4A), the value of P remains nearly the same (squares, FIG. 4E).

[0218] To better understand the retention of PEG-BB within individual cells, the variation in intracellular PEG-BB fluorescence among different cell was quantified. To do so, fluorescent area fraction, Fa, was introduced, which is defined as the fluorescent area of a cell, Af, divided by the cytoplasm area. The cytoplasm area is calculated by subtracting the nuclear area, An, from the cell area, Ac, as illustrated by the inset in FIG. 4F As cells proliferate, Fa not only significantly decreases but also shows a higher extent of variation, as shown by the violin plots in FIG. 4F Note that compared to Day 2, the variation in Fais lower on Day 3. This is likely because on Day 3 the maximal value of Fawithin individual cells is relatively low so that the range of Fabecomes smaller compared to Day 2, resulting in smaller variation in Faon Day 3. Nonetheless, after inhibiting cell proliferation by colchicine, not only there is no significant difference in Favalues but also the variation in Faremains nearly the same across three days, as shown by the violin plots in Fig. 4F. Consistent with this understanding, for the well- differentiated HBECs, PEG-BB remains intracellular after 7 days. Taken together, the results show that the decay of intracellular PEG-BB fluorescence is due to cell proliferation and that PEG-BB molecules remain intracellular for at least 3 days after inhibiting cell proliferation. Cellular uptake of PEG-BB nanocarrier is driven by bottlebrush architecture enhanced endocytosis

[0219] To further explore the role of molecular architecture in the cellular uptake of polymeric carriers, the uptake of FITC labeled 2 MDa dextran by NIH-3T3 fibroblasts was quantified. This dextran has a MW on the same order as PEG-BB but is a randomly branched molecule, a molecular architecture that is qualitatively different from the brush-like PEG-BB. No intracellular fluorescence among all NIH-3T3 fibroblasts was found (FIG. 5A, FIG. S4A). Further decreasing the dextran molecular weight to 70 kDa results in an unmeasurable increase in cellular uptake (FIG. 5B, FIG. S4B); this suggests that the molecular weight of dextran molecules has a negligible effect on their uptake by NIH-3T3 fibroblasts. However, dextran and PEG are of different chemical species, which are known to affect the efficiency of cellular uptake48 50. To this end, the uptake of PEG-LG, which has a comparable MW and hydrodynamic size to PEG-BB but with loosely grafted PEG side chains, was quantified. Despite that the intracellular PEG-LG fluorescence is slightly higher than that of 70 kDa dextran, it is dramatically lower than that of PEG-BB, as shown by the fluorescence images in FIG. 5C and FIG. S4C. Together with the minimum uptake of PEG-LG by HBECs, these results confirm that the bottlebrush architecture enables efficient cellular uptake of PEG-BB polymers.

[0220] The size of PEG-BB is too large to cross the epithelium by diffusion through cell junctions, which typically necessitate very small molecules of 2~4 nm or less51. Alternatively, endocytosis allows a wide range of substances with various sizes and extents of hydrophobicity to traverse the cell membrane52. Given that neither HBECs nor NIH-3T3 fibroblasts are phagocytic cells, it was examined whether the cellular uptake of PEG-BB is regulated by pinocytosis pathways. To explore this, cells with were treated wortmannin, a non-specific inhibitor often considered for macropinocytosis, an endocytosis pathway for nonspecific internalization of large amounts of extracellular fluid53. Upon treating NIH-3T3 fibroblasts with wortmannin, nearly all PEG-BB fluorescence is extracellular, outlining the cell contour, as visualized by the left panel in FIG. 5D and FIG. S5A. Following the removal of free PEG- BB by replacing the medium, negligible fluorescence is detected within NIH-3T3 fibroblasts (right panel in FIG. 5D and FIG. S5A). Yet, recent studies suggest that wortmannin can also impair clathrin- and caveolin-mediated endocytosis54. To identify the specific endocytosis pathways involved in PEG-BB internalization, NIH-3T3 fibroblasts were treated with chlorpromazine which is known to inhibit clathrin-mediated endocytosis55but recently found to impede macropinocytosis50. Minimum cellular uptake of PEG-BB is also observed (FIG. 5E and FIG. S5B). Finally, treating NIH-3T3 fibroblasts with nystatin, an inhibitor of caveolin- mediated endocytosis56, effectively prevents the cellular uptake of PEG-BB (FIG. 5F and Fig. S5C). These results suggest that the internalization of PEG-BB by NIH-3T3 fibroblasts is likely mediated by both macropinocytosis and caveolin-mediated endocytosis.

[0221] Based on the knowledge obtained for the uptake of PEG-BB by NIH-3T3 fibroblasts, the three endocytosis inhibitors were applied to well-differentiated HBECs. As expected, all these three inhibitors significantly reduce the cellular uptake of PEG-BB molecules from both the apical and the basal sides, as shown by the fluorescence images in FIG. 5G-I. Quantitatively, for HBECs treated with inhibitors, the fluorescence area decreases from 32 ± 5% to -3% and from 26 ±6% to -3% for PEG-BB added to the apical and to the basal sides, respectively (FIG. 5J). The results collectively show that that the internalization of PEG-BB by cells is regulated by bottlebrush architecture enhanced endocytosis (FIG. 5K).

[0222] Discussion

[0223] It was discovered that bottlebrush PEG polymers, can rapidly penetrate through the mucus gel and the periciliary layer to translocate across the human airway epithelium via molecular architecture enhanced endocytosis. The PEG-BB is highly anisotropic, featuring a contour length of -250 nm, a cross-section of -20 nm, and a hydrodynamic diameter of -40 nm. The design of PEG-BB draws inspiration from the brush-like mucin biopolymers and mucus-penetrating PEGylated nanoparticles. The size and shape of PEG-BB are based on the mesh sizes of mucus hydrogel (10-100 nm) and periciliary brush layer (20-40 nm). By comparing the cellular uptake of bottlebrush PEG against loosely grafted PEG with a comparable hydrodynamic diameter but a lower grafting density, it was shows that high grafting density is needed to efficient cellular uptake. Further, it was shown that the cellular uptake of PEG-BB is significantly higher than randomly branched dextran molecules regardless of their molecular weight. Manipulating the proliferation of NIH-3T3 fibroblasts reveals that the retention of internalized PEG-BB is determined by cell proliferation rate. Finally, by inhibiting endocytosis pathways, it was shown the uptake of PEG-BB by fibroblasts and well-differentiated HBECs is regulated by endocytosis.

[0224] Compared to existing mucosal delivery systems, the developed PEG-BB serves as a unique nanocarrier in the context of synthesis and design. For example, the ability of the PEGylated nanoparticles to sneak through the sticky mucus hydrogel requires uniform distribution and high grafting density of PEG chains. However, these two parameters are difficult to be precisely controlled because of the nature of the grafting process. Most PEGylated nanoparticles are synthesized using a grafting-through approach, where functional PEG chains are attached to the grafting sites on the surface of the nanoparticle57. At a relatively high grafting density, the already highly grafted chains generate steric hindrance to prevent access to the grafting sites58. This difficulty is further exacerbated for small nanoparticles (<100 nm) with a relatively high surface curvature59. By contrast, PEG-BB is synthesized by polymerizing PEG macromonomers with a prescribed molecular weight (Nsc). Within a PEG- BB molecule, the PEG side chains are evenly distributed and have a precisely controlled grafting density (l / (s + 1)). Moreover, the total molecular weight of PEG-BB, or the number of side chains ( nsc), can be tuned in a wide range through well-established living polymerization techniques. Thus, compared with the PEGylated nanoparticles, the synthesis of PEG-BB is more controlled, enabling prescribed molecular architecture parameters, [Asc, nsc, 5].

[0225] The prescribed molecular architecture parameters offer unprecedented control over the geometry and physical properties of PEG-BB. For instance, using longer side chains and / or increasing grafting density increases the cross-section of the bottlebrush, so that the extent of anisotropy of PEG-BB can be tuned for targeted therapeutic delivery60 63. An example is that using a small number (~30) of relatively long PEG chains (10 kDa) results in a PEG carrier with a spherical geometry, which has been demonstrated to enable efficient in vivo delivery of nucleic acid therapeutics such small interfering RNA (siRNA)64and antisense oligonucleotides65,66. By contrast, in our studies, PEG-BB consists of many (-1000) relatively short PEG chains (1 kDa), exhibiting a highly antistrophic, wormlike geometry. Compared to conventional rigid nanoparticles that can be easily trapped within network meshes67 70, PEG- BB is a flexible, wormlike nanocarrier, enabling rapid transport through gels and extracellular matrices via reptation71. Moreover, drugs for specific diseases can be loaded to and released by PEG-BB using a chemical approach. For instance, multiple kinds of small molecule drugs can be conjugated to macromonomers via cleavable linkers that activate to release drugs, offering strategies for improving monotherapies and combination therapies for multiple myeloma.72Together with the versatility in molecular design, the ability to rapidly penetrate through mucus to be internalized by epithelial cells enables PEG-BB as a precision nanocarrier73for mucosal drug delivery74.

[0226] Bibliography 1. Loira-Pastoriza, C., Todoroff, J. & Vanbever, R. Delivery strategies for sustained drug release in the lungs. Adv. Drug Deliv. Rev. 75, 81-91 (2014).

[0227] 2. Kan, S. et al. Airway epithelial-targeted nanoparticles for asthma therapy. Am. J. Physiol. Lung Cell. Mol. Physiol. 318, L500-L509 (2020).

[0228] 3. Pramanik, S. et al. Nanoparticle-based drug delivery system: The magic bullet for the treatment of chronic pulmonary diseases. Mol. Pharm. 18, 3671-3718 (2021).

[0229] 4. Kuzmov, A. & Minko, T. Nanotechnology approaches for inhalation treatment of lung diseases. J. Control. Release 219, 500-518 (2015).

[0230] 5. Button, B. et al. A peri ciliary brush promotes the lung health by separating the mucus layer from airway epithelia. Science (80-. ). 337, 937-941 (2012).

[0231] 6. Cai, L.-H. Structure and function of airway surface layer of the human lungs & mobility of probe particles in complex fluids. (THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL, 2012).

[0232] 7. Hill, D. B., Button, B., Rubinstein, M. & Boucher, R. C. Physiology and pathophysiology of human airway mucus. Physiol. Rev. 102, 1757-1836 (2022).

[0233] 8. Leal, J., Smyth, H. D. C. & Ghosh, D. Physicochemical properties of mucus and their impact on transmucosal drug delivery. Int. J. Pharm. 532, 555-572 (2017).

[0234] 9. Witten, J., Samad, T. & Ribbeck, K. Selective permeability of mucus barriers. Curr. Opin. BiotechnoL 52, 124-133 (2018).

[0235] 10. Lieleg, O. & Ribbeck, K. Biological hydrogels as selective diffusion barriers. Trends Cell Biol. 21, 543-51 (2011).

[0236] 11. Gizurarson, S. The effect of cilia and the mucociliary clearance on successful drug delivery. Biol. Pharm. Bull. 38, 497-506 (2015).

[0237] 12. Knowles, M. R. & Boucher, R. C. Mucus clearance as a primary innate defense mechanism for mammalian airways. J. Clin. Invest. 109, 571-577 (2002).

[0238] 13. Wang, G. et al. Increasing epithelial junction permeability enhances gene transfer to airway epithelia in vivo. Am. J. Respir. Cell Mol. Biol. 22, 129-138 (2000).

[0239] 14. Ferrari, S., Geddes, D. M. & Alton, E. W. F. W. Barriers to and new approaches for gene therapy and gene delivery in cystic fibrosis. Adv. Drug Deliv. Rev. 54, 1373-1393 (2002).

[0240] 15. Denker, B. M. & Nigam, S. K. Molecular structure and assembly of the tight junction. Am. J. Physiol. - Ren. Physiol. 274, 1-9 (1998).

[0241] 16. Lai, S. K. et al. Rapid transport of large polymeric nanoparticles in fresh undiluted human mucus. Proc. Natl. Acad. Sci. U. S. A. 104, 1482-1487 (2007). 17. Lai, S. K., Wang, Y.-Y. & Hanes, J. Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues. Adv. Drug Deliv. Rev. 61, 158-171 (2009).

[0242] 18. Huckaby, J. T. & Lai, S. K. PEGylation for enhancing nanoparticle diffusion in mucus. Adv. Drug Deliv. Rev. 124, 125-139 (2018).

[0243] 19. Button, B. et al. Roles of mucus adhesion and cohesion in cough clearance. Proc. Natl. Acad. Sci. U. S. A. 115, 12501-12506 (2018).

[0244] 20. Henderson, A. G. et al. Cystic fibrosis airway secretions exhibit mucin hyperconcentration and increased osmotic pressure. J. Clin. Invest. 124, 3047-3060 (2014).

[0245] 21. Pednekar, D. D. et al. From static to dynamic: A review on the role of mucus heterogeneity in particle and microbial transport. ACS Biomater. Sci. Eng. 8, 2825- 2848 (2022).

[0246] 22. Suk, J. S. et al. The penetration of fresh undiluted sputum expectorated by cystic fibrosis patients by non-adhesive polymer nanoparticles. Biomaterials 30, 2591-2597 (2009).

[0247] 23. Schuster, B. S., Suk, J. S., Woodworth, G. F. & Hanes, J. Nanoparticle diffusion in respiratory mucus from humans without lung disease. Biomaterials 34, 3439-3446 (2013).

[0248] 24. Kirch, J. et al. Optical tweezers reveal relationship between microstructure and nanoparticle penetration of pulmonary mucus. Proc. Natl. Acad. Sci. U. S. A. 109, 18355-18360 (2012).

[0249] 25. Wang, Y.-Y. et al. Addressing the PEG mucoadhesivity paradox to engineer nanoparticles that “slip” through the human mucus barrier. Angew. Chemie 120, 9872- 9875 (2008).

[0250] 26. Smart, J. D. The basics and underlying mechanisms of mucoadhesion. Adv. Drug Deliv. Rev. 57, 1556-1568 (2005).

[0251] 27. Fulcher, M. L., Gabriel, S., Burns, K. A., Yankaskas, J. R. & Randell, S. H. Well- differentiated human airway epithelial cell cultures. Methods Mol. Med. 107, 183-206 (2005).

[0252] 28. Leung, C., Wadsworth, S. J., Jasemine Yang, S. & Dorscheid, D. R. Structural and functional variations in human bronchial epithelial cells cultured in air-liquid interface using different growth media. Am. J. Physiol. - Lung Cell. Mol. Physiol. 318, L1063- L1073 (2020).

[0253] 29. Salai, M. et al. The inhibitory effects of colchicine on cell proliferation and mineralisation in culture. J. Bone Jt. Surg. Br. Vol. 83-B, 912-915 (2001).

[0254] 30. Rose, M. C. Mucins: Structure, function, and role in pulmonary diseases. Am. J. Physiol. - Lung Cell. Mol. Physiol. 263, L413-29 (1992).

[0255] 31. Hattrup, C. L. & Gendler, S. J. Structure and function of the cell surface (tethered) mucins. Annu. Rev. Physiol. 7 , 431-457 (2008).

[0256] 32. Thornton, D. J., Rousseau, K. & McGuckin, M. A. Structure and function of the polymeric mucins in airways mucus. Annu. Rev. Physiol. 70, 459-486 (2008).

[0257] 33. Nian, S. et al. Molecular architecture directs linear-bottlebrush-linear triblock copolymers to self-assemble to soft reprocessable elastomers. ACS Macro Lett. 8, 1528-1534 (2019).

[0258] 34. Cai, L.-H. et al. Soft Poly(dimethylsiloxane) elastomers from architecture-driven entanglement free design. Adv. Mater. 27, 5132-5140 (2015).

[0259] 35. Nian, S., Huang, B., Freychet, G., Zhernenkov, M. & Cai, L.-H. H. Unexpected folding of bottlebrush polymers in melts. Macromolecules 56, 2551-2559 (2023).

[0260] 36. Cai, L. H. Molecular understanding for large deformations of soft bottlebrush polymer networks. Soft Matter 16, 6259-6264 (2020).

[0261] 37. Paturej, J. J., Sheiko, S. S., Panyukov, S. & Rubinstein, M. Molecular structure of bottlebrush polymers in melts. Sci. Adv. 2, el601478 (2016).

[0262] 38. Wong, L., Sevimli, S., Zareie, H. M., Davis, T. P. & Bulmus, V. PEGylated functional nanoparticles from a reactive homopolymer scaffold modified by thiol addition chemistry. Macromolecules 43, 5365-5375 (2010).

[0263] 39. Perrier, S. 50th anniversary perspective: RAFT polymerization - A user guide. Macromolecules 50, 7433-7447 (2017).

[0264] 40. Caracciolo, S., Mognetti, B. M. & Pelissetto, A. Polymer size in dilute solutions in the good-solvent regime. J. Chem. Phys. 125, (2006).

[0265] 41. Wu, R., Sato, G. H. & Whitcutt, M. J. Developing differentiated epithelial cell cultures: Airway epithelial cells, in Toxicological Sciences vol. 6 580-590 (1986).

[0266] 42. Pezzulo, A. A. et al. The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia. Am. J. Physiol. - Lung Cell. Mol. Physiol. 300, 25-31 (2011).

[0267] 43. Dvorak, A., Tilley, A. E., Shaykhiev, R., Wang, R. & Crystal, R. G. Do airway epithelium air-liquid cultures represent the in vivo airway epithelium transcriptome? Am. J. Respir. Cell Mol. Biol. 44, 465-473 (2011).

[0268] 44. Baldassi, D., Gabold, B. & Merkel, O. M. Air-liquid interface cultures of the healthy and diseased human respiratory tract: Promises, challenges, and future directions. Adv. NanoBiomed Res. 1, 2000111 (2021).

[0269] 45. He, Z.-J., Chu, C., Dickson, R. & Cai, L.-H. A gel-coated air-liquid-interface culture system with tunable substrate stiffness matching healthy and diseased lung tissues. bioRxiv 2023.07.29.550710 (2023) doi: 10.1101 / 2023.07.29.550710.

[0270] 46. Schneider, C. S. et al. Nanoparticles that do not adhere to mucus provide uniform and long-lasting drug delivery to airways following inhalation. Sci. Adv. 3, (2017).

[0271] 47. Kurek, J. et al. Cytotoxic, analgesic and anti-inflammatory activity of colchicine and its C-10 sulfur containing derivatives. Sci. Rep. 11, 1-12 (2021).

[0272] 48. Hu, Y., Xie, J., Tong, Y. W. & Wang, C. H. Effect of PEG conformation and particle size on the cellular uptake efficiency of nanoparticles with the HepG2 cells. J. Control. Release 118, 7-17 (2007).

[0273] 49. Li, L. et al. The effect of the size of fluorescent dextran on its endocytic pathway. Cell Biol. Int. 39, 531-539 (2015).

[0274] 50. Kamei, N. et al. Optimization of the method for analyzing endocytosis of fluorescently tagged molecules: Impact of incubation in the cell culture medium and cell surface wash with glycine-hydrochloric acid buffer. J. Control. Release 310, 127-140 (2019).

[0275] 51. Talukdar, S., Emdad, L., Das, S. K. & Fisher, P. B. GAP junctions: multifaceted regulators of neuronal differentiation. Tissue Barriers 10, (2022).

[0276] 52. Doherty, G. J. & McMahon, H. T. Mechanisms of endocytosis. Annu. Rev. Biochem. 78, 857-902 (2009).

[0277] 53. Khalil, I. A., Kogure, K., Akita, H. & Harashima, H. Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. Pharmacol. Rev. 58, 32-45 (2006).

[0278] 54. Itagaki, M., Nasu, Y., Sugiyama, C., Nakase, I. & Kamei, N. A universal method to analyze cellular internalization mechanisms via endocytosis without non-specific cross-effects. FASEB J. 37, 1-15 (2023).

[0279] 55. Wang, L. H., Rothberg, K. G. & Anderson, R. G. W. Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J. Cell Biol. 123, 1107-1117 (1993).

[0280] 56. Chen, Y. et al. Cholesterol sequestration by nystatin enhances the uptake and activity of endostatin in endothelium via regulating distinct endocytic pathways. Blood 117, 6392-6403 (2011).

[0281] 57. Li, Y., Kroger, M. & Liu, W. K. Endocytosis of PEGylated nanoparticles accompanied by structural and free energy changes of the grafted polyethylene glycol. Biomaterials 35, 8467-8478 (2014).

[0282] 58. Zou, Y., Rossi, N. A. A., Kizhakkedathu, J. N. & Brooks, D. E. Barrier capacity of hydrophilic polymer brushes to prevent hydrophobic interactions: Effect of graft density and hydrophilicity. Macromolecules 42, 4817-4828 (2009).

[0283] 59. Lin, J., Zhang, H., Morovati, V. & Dargazany, R. PEGylation on mixed monolayer gold nanoparticles: Effect of grafting density, chain length, and surface curvature. J. Colloid Interface Sci. 504, 325-333 (2017).

[0284] 60. Zhang, Z. et al. Length effects of cylindrical polymer brushes on their: In vitro and in vivo properties. Biomater. Sci. 7, 5124-5131 (2019).

[0285] 61. Niederberger, A. et al. Stiffness-dependent intracellular location of cylindrical polymer brushes. Macromol. Rapid Commun. 42, 1-6 (2021).

[0286] 62. Milliner, M. Molecular polymer bottlebrushes in nanomedicine: therapeutic and diagnostic applications. Chem. Commun. 5683-5716 (2022) doi: 10.1039 / d2cc01601j.

[0287] 63. Rabanel, J. M. et al. Deep tissue penetration of bottle-brush polymers via cell capture evasion and fast diffusion. ACS Nano (2022) doi: 10.1021 / acsnano.2cl0554.

[0288] 64. Fang, Y. et al. Inhalable bottlebrush polymer bioconjugates as vectors for efficient pulmonary delivery of oligonucleotides. ACS Nano (2023) doi: 10.1021 / acsnano.3c08660.

[0289] 65. Wang, D. et al. Targeting oncogenic KRAS with molecular brush-conjugated antisense oligonucleotides. Proc. Natl. Acad. Sci. U. S. A. 119, 1-25 (2022).

[0290] 66. Wang, D. et al. Bottlebrush-architectured polyethylene glycol) as an efficient vector for RNA interference in vivo. Sci. Adv. 5, (2019).

[0291] 67. Cai, L.-H. H., Panyukov, S. & Rubinstein, M. Mobility of nonsticky nanoparticles in polymer liquids. Macromolecules 44, 7853-7863 (2011).

[0292] 68. Cai, L.-H., Panyukov, S. & Rubinstein, M. Hopping diffusion of nanoparticles in polymer matrices. Macromolecules 48, 847-862 (2015).

[0293] 69. Ge, T. Scaling perspective on dynamics of nanoparticles in polymers: Length- and time-scale dependent nanoparti cle-polymer coupling. Macromolecules 56, 3809-3837 (2023).

[0294] 70. Wang, J. et al. Diffusion of rod-like nanoparticles in non-adhesive and adhesive porous polymeric gels. J. Meeh. Phys. Solids 112, 431-457 (2018).

[0295] 71. Rubinstein, M. & Colby, R. H. Polymer Physics. (Oxford University Press, Oxford, UK, 2003).

[0296] 72. Detappe, A. et al. Molecular bottlebrush prodrugs as mono- and triplex combination therapies for multiple myeloma. Nat. NanotechnoL 18, 184-192 (2023).

[0297] 73. Mitchell, M. J. et al. Engineering precision nanoparticles for drug delivery. Nat. Rev. DrugDiscov. 20, 101-124 (2021).

[0298] 74. Woodrow, K. A., Bennett, K. M. & Lo, D. D. Mucosal vaccine design and delivery. Annu. Rev. Biomed. Eng. 14, 17-46 (2012).

[0299] 75. Wijmans, C. M. & Zhulina, E. B. Polymer brushes at curved surfaces. Macromolecules 26, 7214-7224 (1993).

[0300] 76. Lee, H., Venable, R. M., MacKerell, A. D. & Pastor, R. W. Molecular dynamics studies of polyethylene oxide and polyethylene glycol: Hydrodynamic radius and shape anisotropy. Biophys. J. 95, 1590-1599 (2008).

[0301] 77. Zhu, J. et al. Digital assembly of spherical viscoelastic bio-ink particles. Adv. Funct. Mater. 32, 1-11 (2022).

[0302] 78. Birshtein, T. M., Borisov, O. V., Zhulina, Y. B., Khokhlov, A. R. & Yurasova, T. A. Conformations of comb-like macromolecules. Polym. Sci. U.S.S.R. 29, 1293-1300 (1987).

[0303] 79. Murat, M. & Grest, G. S. Polymers end-grafted onto a cylindrical surface. Macromolecules 24, 704-708 (1991).

[0304] 80. Dinic, J. & Sharma, V. Flexibility, extensibility, and ratio of Kuhn length to packing length govern the pinching dynamics, coil-stretch transition, and rheology of polymer solutions. Macromolecules 53, 4821-4835 (2020).

[0305] EXAMPLE II

[0306] Molecular structure of a bottlebrush polymer in solution.

[0307] In a bottlebrush polymer, the side chains highly overlap with each other, resulting in steric repulsion so that the side chains extend away from the bottlebrush backbone, forming a cylindrical shape with the cross-section illustrated by FIG. S1A. To calculate the cross-section of the bottlebrush or the size of a side chain, Rsc, the profile of the volume fraction, cf>(r), of the side chains at the distance r from the bottlebrush backbone was considered.

[0308] Here, b is the Kuhn monomer size, g(r) is the number of monomers per correlation blob, ^(r) « bgv(r) is the correlation length, and v is the Flory exponent depending on solvent quality (for theta solvent v = 1 / 2 and for a good or athermal solvent v = 3 / 5)71.

[0309] Within the cylinder-like bottlebrush polymer, the correlation length is related to the linear grafting density 1 / 1 of side chains5,75:

[0310] ^(r) « (rl)1 / 2, for r > b (S3) which increases the distance r by a power of 1 / 2 (FIG. SIB). Note that in the bottlebrush polyethylene glycol (PEG-BB) polymer, the grafting distance is very small with 1 = 0.254 nm, much smaller than the size b = 0.8 nm of a polyethylene glycol (PEG) Kuhn monomer76,77. Thus, at the length scale r < b, the side chains fill the space to form an exclusion zone for solvents, as predicted by the previous theory78and confirmed by simulation79, as well as illustrated by the shadowed circle in FIG. S1A and the dashed line in FIG. SIB. However, the exclusion zone is very small on the order of Kuhn monomer size. Thus, we ignore the effect of exclusion zone was ignored on the bottlebrush thickness and the region with r > b was focused on, where the polymer chains interact with solvent molecules.

[0311] Substituting eq. (S3) to eq. (S2), the volume fraction profile can be re-written in terms of the distance r from the bottlebrush backbone:

[0312] The size of a side chain, Rsc, can be determined based on mass conservation: r Rsc

[0313] Jb where Nk scis the number of Kuhn monomers per side chain. Solving eq. (S4) one obtains:

[0314] The bottlebrush polymer is effectively a ‘fat’ linear polymer with an effective Kuhn length about the cross-section of the bottlebrush, be« 2RSC. The radius gyration of this ‘fat’ linear polymer is proportional to the end-to-end distance: where a = 0.4 for a linear polymer in good solvent40and Lmax= nscl is the contour length of the bottlebrush backbone with nscbeing the number of side chains per bottlebrush. Since water is a good solvent for PEG (v = 3 / 5), the size of the side chain can be rewritten as: f°rgood or athermal solvent (^8)

[0315] This suggests that the side chain size not only increases with the grafting density 1 / 1 but also scales with the polymer MW by a power of 3 / 4, higher than 3 / 5 for an unperturbed linear chain in good solvent.

[0316] For PEG in water, the size and mass of a Kuhn monomer are, respectively, b = 0.8 nm and Mo= 44 g / mol76,80. In the densely grafted PEG-BB, the number of side chains is nsc= 990, the grafting distance is 1 = 0.254 nm, and the number of Kuhn monomers per side chain is Nk,sc=Msc / Mo ~ 22, in which Msc= 950 g / mol is the molecular weight of a PEG side chain. Substituting these numbers into eqs. (S8) and (S7), one obtains the cross-section of the bottlebrush, be« 2RSC« 20 nm, and the size of the bottlebrush, Rg« 36 nm . The hydrodynamic diameter dhof a ‘fat’ linear polymer is related to its radius of gyration Rgby dh= 1.25Rg« 45 nm (Table 8.4 in ref.71). Considering that scaling theory ignores prefactors on the order of unity, the predicted value agrees reasonably well with the measured hydrodynamic diameter 37 ± 0.4 nm (FIG. IF).

[0317] For the loosely grafted PEG (PEG-LG), the number of side chains is nsc= 750 and the grafting distance 1 = 1.524 nm is much larger than the size of a PEG Kuhn monomer. As a result, the side chains are not much overlapped and adopt a nearly unperturbed conformation. Therefore, the backbone of the bottlebrush polymer is not strained and adopts a self-avoiding random walk with Rg« ab(Lmax / b)3 / 5« 33 nm (where b « 1.7 nm for a methyl methacrylate-based polymer35and Lmax« 1100 nm is the backbone contour length).

[0318] Bibliography

[0319] 1. Rubinstein, M. & Colby, R. H. Polymer Physics. (Oxford University Press, 2003).

[0320] 2. Wijmans, C. M. & Zhulina, E. B. Polymer brushes at curved surfaces. Macromolecules 26, 7214-7224 (1993).

[0321] 3. Button, B. et al. A periciliary brush promotes the lung health by separating the mucus layer from airway epithelia. Science 337, 937-941 (2012).

[0322] 4. Lee, H., Venable, R. M., MacKerell, A. D. & Pastor, R. W. Molecular dynamics studies of polyethylene oxide and polyethylene glycol: Hydrodynamic radius and shape anisotropy. Biophys J 95, 1590-1599 (2008). 5. Zhu, J. et al. Digital assembly of spherical viscoelastic bio-ink particles. Adv Funct Mater 32, 1-11 (2022).

[0323] 6. Birshtein, T. M., Borisov, O. V, Zhulina, Y. B., Khokhlov, A. R. & Yurasova, T. A. Conformations of comb-like macromolecules. Polym Set USSR 29, 1293-1300 (1987).

[0324] 7. Murat, M. & Grest, G. S. Polymers end-grafted onto a cylindrical surface. Macromolecules 24, 704-708 (1991).

[0325] 8. Caracciolo, S., Mognetti, B. M. & Pelissetto, A. Polymer size in dilute solutions in the good-solvent regime. J Chem Phys 125, (2006).

[0326] 9. Dinic, J. & Sharma, V. Flexibility, extensibility, and ratio of Kuhn length to packing length govern the pinching dynamics, coil-stretch transition, and rheology of polymer solutions. Macromolecules 53, 4821-4835 (2020).

[0327] 10. Nian, S., Huang, B., Freychet, G., Zhemenkov, M. & Cai, L.-H. H. Unexpected folding of bottlebrush polymers in melts. Macromolecules 56, 2551-2559 (2023).

[0328] All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification, this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details herein may be varied considerably without departing from the basic principles of the invention.

Claims

WHAT IS CLAIMED IS:

1. A bottlebrush polyethylene glycol (PEG-BB) nanocarrier of polyethylene glycol (PEG) macromonomers forming a linear backbone grafted by PEG side chains, wherein the backbone has between about 750 to about 1200 side chains extending radially away from the backbone.

2. The nanocarrier of claim 1 , wherein the PEG macromonomers have a molecular weight of about 200 grams / mole (g / mol) to about 1500 g / mol.

3. The nanocarrier of claim 2, wherein the PEG macromonomers have a molecular weight of about 400 grams / mole (g / mol) to about 1200 g / mol.

4. The nanocarrier of claim 2, wherein the PEG macromonomers have a molecular weight of about 1000 g / mol.

5. The nanocarrier of claim 1 , wherein the PEG macromonomers have a molecular weight of about 950 g / mol.

6. The nanocarrier of claim 1, wherein the PEG macromonomers are methacrylate- terminated.

7. The nanocarrier of claim 1, wherein the grafting density of the PEG side chains is about 0.6 to about 4 side chains per nanometer as measured by 'H NMR.

8. The nanocarrier of claim 1, wherein the grafting density of the PEG side chains is about 4 side chains per nanometer (4 nm’1) as measured by 'H NMR.

9. The nanocarrier of claim 1, wherein there are no spacers in the backbone.

10. The nanocarrier of claim 1, wherein PEG-BB has a contour length of about 150-350 nm, a cross-section of about 15-30 nm, and a hydrodynamic diameter of about 30-50 nm.

11. The nanocarrier of claim 1, wherein PEG-BB has a contour length of about 250 nm, a cross-section of about 20 nm, and a hydrodynamic diameter of about 40 nm.

12. The nanocarrier of any one of claims 1 to 11 further comprising one or more therapeutic, prophylactic, or diagnostic agents.

13. A pharmaceutical composition comprising the nanocarrier of claims 12 and one or more pharmaceutically acceptable carriers.

14. A method of administering one or more therapeutic, prophylactic, and / or diagnostic agents to a subject in need thereof, the method comprising administering an effective amount of the nanocarrier of claim 12.

15. A method of administering one or more therapeutic, prophylactic, and / or diagnostic agents to airway epithelium cells of a subject in need thereof, the method comprising administering an effective amount of the nanocarrier of claim 12.

16. The method of claim 14, wherein the nanocarrier is administered enterally, parenterally, or topically.

17. The method of claim 14, wherein the nanocarrier is administered to a pulmonary tract.

18. A method to treat a respiratory or mucus disease or disorder comprising administering the nanocarrier of claim 12.

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