Compositions and methods to treat mucus-related diseases

Abstract

Pharmaceutical compositions which either induce transient epithelial-mesenchymal transition (EMT) in epithelial cells, or inhibit fibroblast and immune cell migration, in the treatment of diseases or conditions associated with build-up of viscous extracellular fluids. Delivery vehicles include the use of mucus-penetrating nanoparticles

Description

COMPOSITIONS AND METHODS TO TREAT MUCUS-RELATED DISEASES The present application claims the benefit of U.S. provisional application no.63 / 528,626 filed July 24, 2023, which is incorporated by reference herein in its entirety. BACKGROUND

[0001] It has been extensively demonstrated that cellular behaviors are governed not just by biochemical signaling, but also by biophysical cues (1). For example, stiff substrates can steer stem cells towards osteogenic differentiation (2) and can prompt otherwise non- invasive tumor cells to invade (3). Local topography can determine cell morphology (4) and direct cell migration (5, 6). Cells are capable of responding to externally applied forces, such as stretching and shearing, by rearranging their cytoskeleton and adjusting gene expression (7–10). One biophysical component of the cellular microenvironment that has received relatively little attention, however, is the viscosity of extracellular fluid (ECF). Cells in vivo are often immersed in fluids with viscosities orders of magnitude higher than that of culture medium (11–14) – secreted mucins make airway mucus and saliva viscous (12), excess fibrinogen can raise blood viscosity(13), and hyaluronic acids contribute to the viscosity of interstitial fluids (14). Moreover, abnormal ECF viscosity has been implicated in various diseases, including cystic fibrosis (12) and cancer (11, 15–17). Despite the evidence suggesting a physiological role of ECF viscosity, there has been limited research addressing the subject, and no conclusive trend has been established in the few pioneering studies. In two studies, viscous fluid was found to decrease motility in non-adherent cells or cells moving through fluid in three dimensions (18, 19). In another, there was no change in the motility of adherent keratocytes in viscous ECF (20). SUMMARY

[0002] Embodiments are directed to compositions in the treatment of injuries, wounds, or diseases and disorders associate with a build up of extracellular fluids such as Cystic Fibrosis. Methods of identifying candidate therapeutic agents include assays that measure cellular responses in culture in response to increases in viscosity of the medium.

[0003] In certain aspects, a method of treating a wound, injury, disease or disorder associated with an increase in viscosity of extracellular fluid (ECF) comprises: administering to a147632574.1subject in need of treatment, a composition comprising a therapeutically effective amount of one or more agents, wherein the agents inhibit fibroblast cell motility, immune cell motility, induce transient epithelial-mesenchymal transition (EMT) or combinations thereof. In certain embodiments, the one or more agents comprise small molecule compounds, nucleotide-based antisense reagents, antibodies, enzymes, peptides, organic or inorganic molecules, natural or synthetic compounds. In certain embodiments, the one or more agents modulate cellular ruffling. In certain embodiments, the one or more agents modulating cellular ruffling decrease total cofilin concentration in a cell as compared to a normal control.

[0004] In one preferred aspect, one or more therapeutic agents are administered to a subject with mucus-penetrating liposomes (MPLs). In preferred aspects, MPLs utilized in the present methods and compositions comprise surface-modified liposomes preferably capable of escaping the adhesive and steric interactions with mucin molecules, and penetrating the mucus effectively.

[0005] Preferred MPLs can deliver one or more therapeutic agents to tissue underneath the mucus. In one aspect, an MPL may include polyethylene glycol (PEG) including as a surface layer, for example with low-molecular-weight (<5 kDa) polyethylene glycol (PEG) grafted or otherwise coated onto the liposome surface.

[0006] In a further aspect, preferred MPLs can comprise a linked targeting molecule, e.g. one or more conjugated antibodies, including a conjugated antibody that targets cell-type markers of fibroblasts (FAP), neutrophils / macrophages (CD11), and epithelial cells (E-cadherin).

[0007] In certain aspects, one or more one or more therapeutic agents can be admixed with lipids during MPL synthesis which can provide thereby the therapeutic agent(s) encapsulated in an MPL.

[0008] In certain embodiments, the one or more agents modulating cellular ruffling increase activated cofilin in a cell as compared to a normal control. In certain embodiments, wherein the activated cofilin is increased by administration of a LIM kinase (LIMK) inhibitor. In certain embodiments, wherein the one or more agents modulating cellular ruffling inhibit one or more formin family members. In certain embodiments, a formin family member comprises Dia1.

[0009] In certain embodiments, the one or more agents modulating cellular ruffling comprise inhibitors of YAP nuclear translocation. In certain embodiments, an inhibitor of YAP nuclear translocation comprises YAP / TAZ inhibitor-1, histone deacetylases (HDACs) inhibitors,YAP-binding molecules, quinolinols, dopamine receptor D1 (DRD1) agonists or combinations thereof. In certain embodiments, the one or more agents induce re-epithelization, inhibit mucus triggered fibroblast activation, attenuate immune cell inflammatory responses or combinations thereof. In certain embodiments, the one or more agents modulate intracellular integrins, focal adhesion formation, actin polymerization or combinations thereof.

[0010] In certain embodiments, modulators of integrins comprise: α5β1 integrin agonist-1 (C24H26FN5O9), αvβ1 integrin-IN-1 TFA (C28H35F3N6O8S), integrin-IN-2 (C27H3ON4O3),,integrin Antagonist 27 (C24H2ON4O5).

[0011] In certain embodiments, one or more a4 integrin inhibitors may be utilized as a therapeutic agent in the present methods and compositions, for example natalizumab or an agent disclosed in WO2006-52962.

[0012] In certain embodiments, one or more a9 integrin inhibitors may be utilized as a therapeutic agent in the present methods and compositions.

[0013] In certain embodiments, one or more integrin β4 (ITGB4) inhibitors may be utilized as a therapeutic agent in the present methods and compositions.

[0014] In certain embodiments, the one or more agents are functionalized for one or more of: (i) mucus penetration, (ii) cell type-specific targeting, and (iii) molecules differentially treating epithelial cells, fibroblasts, or immune cells.

[0015] In certain embodiments, the one or more agents are encapsulated in a liposome, a microparticle or nanoparticle. In certain embodiments, the liposomes, microparticles or nanoparticles are mucus-penetrating. In certain embodiments, the liposomes, microparticles or nanoparticles comprise one or more binding domains which specifically bind to one or moretarget cells. In certain embodiments, the one or more binding domains comprise antibodies, antibody fragments, Fab, Fab’, F(ab’)2 fragments, Fv fragments, single chain antibodies, humanized antibodies and antibody fragments, camelized antibodies and antibody fragments, antibody mimetics, DARPins, affibody molecules, affilins, affitins, anticalins, avimers, fynomers, Kunitz domain peptides, monobodies, diabodies, tribodies, peptoids, aptamers, receptor fragments, ligands, oligosaccharides or combinations thereof.

[0016] In certain embodiments, the disease or disorder associated with an increase in viscosity of ECF comprise: cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), tumor microenvironments, lung inflammation, asthma, autoimmune diseases or combinations thereof.

[0017] In certain embodiments, extracellular fluids (ECFs) comprise mucus, interstitial fluids, saliva, cerebrospinal fluids, synovial fluids, pleural fluids, pericardial fluids, peritoneal fluids, plasma or combinations thereof.

[0018] In certain embodiments, the method of treating a subject further comprises combination therapies comprising administering an effective amount of one or more secondary therapeutic agents. In certain embodiments, wherein the one or more secondary therapeutic agents comprise anti-inflammatory agents, cytokines, modulators of checkpoint inhibitors, ligands of checkpoint inhibitors, an agonist, an antagonist, an antibody, an antigen-binding fragment, a polynucleotide, a peptide or combinations thereof.

[0019] In another aspect, a method of identifying candidate therapeutic agents which modulate increase in viscosity of extracellular fluid (ECF) comprises: incubating a cell in media having varying viscosities, contacting the cell with one or more agents, assaying cellular responses to the one or more agents, and identifying a candidate therapeutic agent. In certain embodiments, the cell is an immune cell, an epithelial cell, a fibroblast, or combinations thereof. In certain embodiments, the cellular responses comprise: ruffling of lamellipodia, spreading, focal adhesion formation, migration, or combinations thereof. In certain embodiments, the method is a high-throughput screening method.

[0020] Definitions

[0021] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, and biochemistry).

[0022] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0023] As used in this specification and the appended claims, the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0024] The term “about” is used herein to mean a value that is ±10% of the recited value.

[0025] As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, in reference to defined or described elements of an item, composition, apparatus, method, process, system, etc. are meant to be inclusive or open ended, permitting additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements--or, as appropriate, equivalents thereof--and that other elements can be included and still fall within the scope / definition of the defined item, composition, apparatus, method, process, system, etc.

[0026] As used herein, the term “immune modulator” refers to an agent that acts on a target and regulates the immune system. Non-limiting examples of immune modulators include agents such as, modulators of checkpoint inhibitors, ligands of checkpoint inhibitors, cytokines, derivatives thereof, or any combination thereof. The immune modulator can also include an agonist, an antagonist, an antibody, an antigen-binding fragment, a polynucleotide, such as siRNA, antisense oligonucleotide, a phosphorodiamidate morpholino oligomer (PMO), a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), miRNA, lncRNA, mRNA DNA, or a small molecule.

[0027] “Liposome,” as used herein, generally refers to a particle that may comprise lipids and having a diameter, such as an average diameter, from about 10 nm up to but not includingabout 1 micron, preferably from 100 nm to about 1 micron. The particles can have any shape. Liposomes having a spherical shape are generally referred to as “nanospheres”.

[0028] “Microparticle,” as used herein, generally refers to a particle having a diameter, such as an average diameter, from about 0.2 micron to about 100 microns, preferably from about 0.2 micron to about 50 microns, more preferably from about 0.2 to about 30 microns. The microparticles can have any shape. Microparticles having a spherical shape are generally referred to as “microspheres”.

[0029] As used herein, the term “modulate” includes to “increase” or “decrease” one or more quantifiable parameters, optionally by a defined and / or statistically significant amount. By “increase” or “increasing,” “enhance” or “enhancing,” or “stimulate” or “stimulating,” refers generally to the ability of one or more vesicle-based compositions in accordance with the present disclosure to produce or cause a greater physiological response (i.e., downstream effects) in a cell or in a subject relative to the response caused by either no vesicle-based composition or a control compound. Relevant physiological or cellular responses (in vivo or in vitro) upon administration of vesicle-based compositions will be apparent to persons skilled in the art. An “increased” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more times (e.g., 500, 1000 times), including all integers and decimal points in between and above 1 (e.g., 1.5, 1.6, 1.7. 1.8), the amount produced by no vesicle-based composition (the absence of a bioactive agent) or a control compound. The term “reduce” or “inhibit” may relate generally to the ability of one or more vesicle-based compositions to “decrease” a relevant physiological or cellular response, such as a symptom of a disease or condition described herein, as measured according to routine techniques in the diagnostic art. Relevant physiological or cellular responses (in vivo or in vitro) will be apparent to persons skilled in the art, and may include reductions in the symptoms or pathology of a disease, illness or condition, such as cancer, inflammation or pain. A “decrease” in a response may be “statistically significant” as compared to the response produced by no vesicle-based composition or a control composition, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease, including all integers in between.

[0030] As used herein, the term “subject” or the phrase “a subject in need thereof” refers to any human or non-human animal requiring or desirous of a pharmacological change. For example, a subject in need thereof may be a human patient clinically diagnosed with a disease, such as, for example, cystic fibrosis, and is thus in need of, or is at least desirous of receiving some sort of treatment. In some cases, the methods of the disclosure 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, and primates.

[0031] As used herein, the term “treatment” of a subject (e.g., a mammal, such as a human) or a cell, is any type of intervention used in an attempt to alter the natural course of the subject or cell. Treatment includes, but is not limited to, administration of a vesicle-based composition in accordance with the present disclosure and may be performed either prophylactically or subsequent to the initiation of a pathologic event or contact with an etiologic agent. Also included are “prophylactic” treatments, which can be directed to reducing the rate of progression of the disease or condition being treated, delaying the onset of that disease or condition, or reducing the severity of its onset. “Treatment” or “prophylaxis” does not necessarily indicate complete eradication, cure, or prevention of the disease or condition, or associated symptoms thereof.

[0032] As used herein, the term “therapeutically effective amount” refers to a minimum dosage of a composition in accordance with the present disclosure that provides a desired effect. Therefore, a therapeutically effective amount varies by subject, dosage form / concentration, and results desired. For example, a therapeutically effective amount of an inhalable liquid or aerosol to treat a CF or COPD, might be on the order of micrograms or milligrams per day. In other examples, a therapeutically effective amount of a nasal spray composition, might be on the order of 3 sprays per day per nostril, with each spray about 0.1 mL.

[0033] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0035] FIGS.1A-1H demonstrate that elevated viscosity enhances single cell motility and induces graded, reversible cell spreading. FIG.1A: After 2 hours incubation in media of different viscosities, MDA-MB-231 cells showed a graded increase in cell motility. Error bars: SD; p values were calculated with respect to DMEM, where p ≥ 0.05 (n.s.), < 0.001 (***), < 0.0001; N = 110 (DMEM), 136 (0.25% MC), 136 (0.5% MC), 172 (0.75% MC), 129 (1% MC) 92 (1.5% MC), 84 (2% MC). FIG.1B: Upon changing medium to 1% MC, cells sped up and reached a new steady- state speed within 30 minutes; medium changed at 180 minutes (indicated by an arrow). Envelopes: SD; N= 43 (DMEM), 47 (1% MC). FIGS.1C, 1D: Viscous media made with different polymers increased cell motility, but non-viscous dextran medium with similar osmolarity to other viscous media did not for MDA-MB-231 cells (FIG.1C) or NIH 3T3 cells (FIG.1D); measurements were performed after overnight incubation. Error bars: SD; p values were calculated with respect to DMEM, where p ≥ 0.05 (n.s.), < 0.0001 (****); for c) N = 293 (DMEM), 393 (1% MC), 176 (1% PEO), 75 (1% dextran), for FIG.1D N = 89 (DMEM), 120 (1% MC), 102 (1%PEO), 114 (1% dextran). FIG.1E: Cells started spreading immediately upon the addition of 1% MC at 05:30. Left: eGFP-F-tractin, center: interference reflection microscopy (IRM), right: brightfield (BF); timestamp: mm:ss; scale: 20 μm. FIG.1F: The increase in cell spread area plateaued approximately 20 minutes after medium change, with the final degree of spreading depending on the degree of viscosity. Envelopes: SEM; N = 19 (0.5% MC), 25 (0.75% MC), 16 (1% MC) 15 (1.5% MC), 20 (2% MC). FIG.1G: Viscosity-induced increases in spread area were reversed by replacing 1% MC with DMEM. Envelope: SD; N = 19. FIG.1H: Increases in spread area and speed in response to viscosity changes werecorrelated; R2= 0.84.

[0036] FIGS.2A-2H demonstrate that membrane ruffling is critical for the cellular responses to viscosity changes, and is inversely correlated with viscosity-dependent cell spreading. FIG.2A: MDA-MB-231 cells are shown just before [i] and 1 minute after [ii] the addition of 2% MC. Note that only actively ruffling areas (red arrowheads) and not areas devoid of ruffles (white arrowheads) spread in response to viscous medium. Scale: 20 μm. FIG.2B: Viscous medium added at 15 minutes suppressed membrane ruffling, and higher viscosity led to more complete and more rapid suppression. Representative images of eGFP-F-tractin and segmented ruffles (red) are shown in the inset. Envelopes: SD; N = 12 (0.5% MC), 11 (1% MC), 14 (2% MC). FIG.2C: Membrane ruffling and cell spreading exhibit an inverse relationship,and a decrease in membrane ruffling immediately precedes an increase in spread area. Envelopes: SEM; N = 8. FIG.2D: A representative image of an MDA-MB-231 cell shows that membrane ruffling was suppressed in 2% MC, but recovered after the viscous medium was washed out. Cell contour traced in red; scale: 20 μm. FIGS.2E, 2F: Fish keratocytes, typically devoid of ruffles even in regular culture medium, did not exhibit increases in speed (FIG.2E) speed or spread area (FIG.2F) when immersed in viscous medium. Error bars: SD; p ≥ 0.05 (n.s.); FIG.2E: N = 28 (RPMI), 23 (1% MC), FIG.2F: N = 17. FIGS.2G, 2H: MDA-MB-231 cells treated with WGA to suppress ruffling did not exhibit increases in g) spread area (FIG.2G) or speed (FIG.2H) in 1% MC. FIG.2G: Envelopes: SD; N = 8. FIG.2H: Error bars: SD; p ≥ 0.05 (n.s.); N = 53 (DMEM + WGA), 59 (1% MC + WGA).

[0037] FIGS.3A-3G demonstrate that viscosity-dependent spreading is mediated by integrin. FIG.3A: In the absence of ECM proteins for integrin binding, cells did not spread in response to 1% MC, which was added at 20 minutes. Envelopes: SEM; N = 16 (1% MC), 21 (1% MC, no ECM). FIG.3B: A representative image of MDA-MB-231 cells shows extensive FA growth and rapid FA assembly and disassembly after the addition of 1% MC at 11 minutes. Timestamp: mm:ss; scale: 40 μm. FIG.3C: The difference in intensity of talin signal between subsequent frames shows significant redistribution of talin immediately following the addition of 1% MC, suggesting extensive nascent adhesion growth. Envelope: SD; N = 9. FIG.3D: FA assembly and disassembly rates increased significantly in 1% MC. Error bars: SD; p < 0.0001 (****); FA data were collected from 9 cells and pooled, with N = 196 (assembly, DMEM), 479 (assembly, 1% MC), 251 (disassembly, DMEM), 308 (disassembly, 1% MC). FIG.3E: Integrated FA area per cell over time shows a decrease followed by partial recovery in adhesion area after the addition of 1% MC at 10 minutes. Envelope: SD; N = 20 cells. FIG.3F: Average individual FA area decreased by 30% in 1% MC. FIG.3G: The total number of FAs per cell per frame increased slightly in 1% MC. FIGS.3G, 3F: Error bars: SD; p < 0.01 (**), < 0.0001 (****); FA data were collected from 20 cells for 20 frames and pooled, with N = 364 (DMEM), 725 (1% MC).

[0038] FIGS.4A-4E demonstrate that actin polymerization is required for the cellular response to elevated viscosity, but myosin II contractility is not. FIG.4A: Representative timelapse images show that latA-treated MDA-MB-231 cells did not exhibit significant increases in spread area after changing medium to 1% MC, unlike untreated cells in Figure 2a.Timestamp: mm:ss; scale: 20 μm. FIG.4B: Viscosity-induced spreading was significantly attenuated after latA treatment. Envelope: SEM; N = 16 (1% MC), 15 (1% MC, latA). FIG.4C Representative kymographs show that retrograde flow was slower in 1% MC. eGFP-F-tractin scale: 20 μm; kymograph scale: t = 45 sec, x = 2 μm. F I G . 4 D : Analysis of kymographs revealed the retrograde flow decreased 10-fold in 1% MC. Error bars: SD; p < 0.0001 (****); N = 37 (DMEM), 27 (1% MC). F I G . 4 E: Blebbistatin-treated cells still spread robustly in 1% MC. Envelopes: SEM; N = 16 (1% MC), 17 (1% MC, blebbistatin).

[0039] FIGS.5A-5I demonstrate that cells generate stronger forces at high viscosity independent of myosin II activity. FIGS.5A, 5B: Representative images of MDA-MB-231 cells show that traction forces increased in 1% MC regardless of whether or not myosin II activity was inhibited by blebbistatin. FIG.5C: Quantification shows that total traction forces increased approximately by 2 fold in 1% MC. Error bars: SD; N = 18 for each condition. FIG.5D: Single- molecule force measurement using TGT revealed extensive engagement of integrin molecules transmitting forces > 54 pN as cells spread in response to elevated viscosity. Color indicates the time of TGT rupture; scale: 20 μm. FIG.5E: TGT ruptures over time increased by 2.4-fold after the addition of 1% MC. Error bars: SD; p < 0.001 (***); N = 8. FIG.5F: Inhibition of myosin II contractility by blebbistatin did not inhibit the cell speed increase in 1% MC. Error bars: SD; p ≥ 0.05 (n.s.), < 0.0001 (****); N = 293 (DMEM), 393 (1% MC), 47 (DMEM, blebbistatin), 39 (1% MC, blebbistatin). FIG.5G: The phase diagram generated by the model after inputting experimental measurements shows that, given the observations that 1) FA assembly / disassembly increase, 2) protrusion increases, and 3) FA area decreases, cortical tension is predicted to decrease as spread area increases upon viscosity change where the protrusion distance was used to infer the extent of cell spreading. FIG.5H: Maximal and average drag forces on a ruffling membrane in DMEM and in 1% MC were estimated using finite element analysis, and the results showed that drag forces increased by two orders of magnitude in 1% MC. FIG.5I: We propose that membrane ruffling is a mechanosensor of ECF viscosity.

[0040] FIGS.6A, 6B show measurements of viscous solutions using a rheometer. FIG. 6A: Viscosity vs. shear curves for various media used in this study. FIG.6B: Viscosity of eachsolution measured at 37oC at 100 Hz shear.

[0041] FIGS.7A, 7B demonstrate that viscous medium does not significantly affect cell persistence in FIG.7A: MDA-MB-231 cells or in FIG.7B: NIH 3T3 fibroblasts. Direction autocorrelation was calculated using DiPer(69); in FIG.7A) N = 305, 370, and 176 for DMEM, 1% MC, and 1% PEO, respectively; in FIG.7B) N = 120, 120, and 102 for DMEM, 1% MC, and 1% PEO, respectively; error bars: SEM.

[0042] FIGS.8A, 8B demonstrate that viscous medium induced speed increases across cell types. FIG.8A: Treating RAW 264.7 macrophages with 1% MC led to an increase in cell speed. Error bars: SD; p < 0.0001 (****); N = 37 (DMEM), 51 (1% MC). FIG.8B: Treating AG19642 non-wound healing human fibroblasts with 1% MC led to an increase in cell speed. Error bars: SD; p < 0.001 (***); N = 64 (DMEM), 57 (1% MC).

[0043] FIGS.9A-9F demonstrate that cells became flatter as they spread in viscous medium. FIG.9A: Timelapse xz-slices showed MDA-MB-231 cells, labelled with eGFP-F- tractin, flattening in viscous medium. Time in mm:ss; 1% MC added at 1 minute; scale: 10 μm. FIG.9B: Cell height was measured 4 μm towards the cell center from the leading edge. Error bars: SD; p ≥ 0.05 (n.s.), < 0.01 (**); N = 4 cells. FIG.9C: Representative images of an MDA- MB-231 nucleus xz-cross-section stained with Hoescht showing nuclear shape change in viscous medium; 1% MC added at 1 minute; scale: 10 μm. FIG.9D: Nuclear height decreased, FIG. 9E: nuclear width increased, and FIG.9F: nuclear volume decreased slightly after MDA-MB- 231 cells were treated with 1% MC at 1 minute; error bars: SD; significance reported with respect to DMEM group; p ≥ 0.05 (n.s.), < 0.1 (*), < 0.01 (**), < 0.001 (***), < 0.0001 (****); N = 12 cells.

[0044] FIG.10 shows representative images of an MDA- MB-231 cell on a PLL-coated substrate show no spreading following the addition of 1% MC. Scale: 20 μm.

[0045] FIGS.11A, 11B demonstrate that both the maximum traction stresses (FIG.11A) and total traction forces (FIG.11B) increased in 1% MC for both untreated and blebbistatin- treated cells. Error bars: SD; p ≥ 0.05 (n.s.), < 0.05 (*); < 0.01 (**); N = 15 cells for each condition.

[0046] FIG.12 shows representative SIM images of myosin IIA staining in MDA-MB- 231 cells after 30 minutes incubation in 1% MC show the formation of myosin stacks. Scale: 10 μm.

[0047] FIGS.13A-13E are images showing that laser ablation decreased cortical tension upon addition of viscous medium followed by a return to baseline. Both the FIG.13A) initial cut distance, and FIG.13B) retraction rate showed a decrease and then recovery in 1% MC. Time refers to duration of incubation in 1% MC; error bars: SD; p ≥0.05 (n.s.), < 0.05 (*); < 0.001 (***); N = 19 (control), 6 (0-5 min), 9 (5-15 min), 25 (>15 min). FIG.13C: The time courses of membrane ruffling and cortical tension of cells subjected to 1% MC reveal that ruffle disappearance began instantaneously upon medium change, but a significant reduction in cortical tension occurred only after a 10-minute delay. FIG.13D: Representative cell images and FIG. 13E) zoomed-in regions show actin arc retraction following laser ablation; white squares highlight ablated regions; ablations performed at 2 seconds; scale: FIG.13D) 20 μm, FIG.13) 10 μm.

[0048] FIGS.14A-14E are simulations of protruding and ruffling lamellipodia show increased drag in viscous medium. FIG.14A: Ruffles in 1% MC experience drag two orders of magnitude greater than ruffles in regular medium. Ruffling was modeled as a periodic behavior with a 20-second period starting with the cell membrane at the leading edge detaching from the substrate to which it was adhered. FIGS.14B, 14C: Maximal and average drag forces on a protruding lamellipodia in DMEM and in 1% MC were estimated using finite element analysis. FIG.14D: Force decomposition shows that the force generated by actin to drive protrusion,FProtrusion, and the retrograde forces of contractility and tension that ultimately lead to ruffling,FRetrograde, are oriented almost exclusively in the xy-plane; therefore FRuffle, the z-oriented component of FRetrograde directly responsible for initiating ruffles, is extremely small. FIG. 14E: In the lamellipodium, the force required to cause buckling increases proportionally with the coefficient of viscous drag. In highly viscous medium cells are incapable of generating the greatly increased forces required to cause buckling to the same extent as in regular medium; instead, lamellipodial buckling is significantly reduced.

[0049] FIGS.15A-15C demonstrate that MDA-MB-231 cells rapidly spread in response to increased viscosity despite osmotic shocks and blocking endocytosis to affect membrane availability. FIG.15A: Cells treated with 80 μM dynasore to suppress endocytosis underwent rapid spread area increases when exposed to 1% MC. Envelope: SEM; N = 22. FIG.15B: Cells treated with hypertonic DMEM underwent rapid spread area increases when exposed to hypertonic 1% MC. Upon subjecting cells to the hypertonic treatment, we observed no increasein the spread area while cells continued to ruffle. This result demonstrates that increasing available membrane stored in caveolae does not increase the spread area. We then immersed the hypertonically treated cells with medium which was both hypertonic (530 mOsm) and viscous. We observed a rapid increase in cell spread area, similar to the cells immersed in isotonic 1% MC, accompanied by decrease in ruffles. This observation indicates the specific membrane reservoir associated with ruffles, contributes to the increase in spread area after viscosity increase is detected by the cell. Envelope: SEM; N = 23. FIG.15C: Cell spreading in 1% MC was slightly curtailed following hypotonic treatment; however, spreading increased significantly when cells were exposed to 2% MC. We observed a relatively slower increase in cell spread area compared to cells immersed in isotonic 1% MC medium. The slower increase in cell spread area was likely due to the elevated membrane tension as a result of osmotic swelling. The observation that the spread area was still increased, though slower, demonstrates that membrane tension modulates the dynamics of cell spreading but does not dictate spreading. Notably, when we immersed the hypotonically treated cells with hypotonic 2% MC, instead of hypotonic 1% MC, we observed larger and more rapid increase in cell spread area. Envelopes: SEM; N = 27 (hypotonic 1% MC), 27 (hypotonic 2% MC).

[0050] FIG.16: To quantify membrane ruffling, confocal z-stacks of fluorescent F- tractin were acquired at 1-minute intervals. The summed projection of these stacks created a strong signal in the tall, actin-rich ruffles, and this signal became weaker as the ruffles changed location or flattened. For each projection, a threshold was manually selected to include membrane ruffling across all frames while excluding other actin structures. The ruffle index (arbitrary unit) was then generated by integrating the thresholded area and normalizing each frame to the mean ruffle area of all the frames captured in DMEM.

[0051] FIG.17 is a schematic diagram depicting the interactions between model components.

[0052] FIG.18 is a schematic of a preparation protocol of drug-encapsulated mucus- penetrating liposomes. DETAILED DESCRIPTION

[0053] Viscous extracellular fluid (ECF), including mucus, interstitial fluid, and saliva are often subjected to variations in viscosity, due to lymph circulation, disease development, and fluctuations in protein secretion. For example, elevated mucus viscosity is associated with cysticfibrosis (CF), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF); increased viscosity is detected in the interstitial fluid of tumor microenvironment. Moreover, aging is associated with elevated fibrinogen and albumin concentrations, increasing the viscosity of plasma and interstitial fluid. Yet the elevated viscosity has been considered a consequence of the diseases, and only is regarded as a symptom of these disease rather than a contributing factor that result in the pathology. As discussed in detail in the examples section, the efforts in characterizing cellular responses to changing fluid viscosity revealed that the common belief can be wrong. In particular, it was found that mesenchymal cell types (e.g. fibroblasts) are equipped with a viscosity sensor and respond to elevated ECF viscosity, counterintuitively migrating nearly twice as fast in more viscous medium. This observation comes with a profound implication: ECF viscosity in the tumor microenvironment might accelerate metastasis because cancer cells migrate at faster speed.

[0054] Furthermore, viscosity-dependent, enhanced migration of neutrophils and macrophages can contribute to prevalent infiltration into the tissues, resulting in systematic low- grade inflammation in geriatric patients. In contrast, it was observed herein, that collective migration of epithelial cells, which lack of viscosity sensor, slows down as the viscosity increases. Using an in vitro model consisting of cocultured fibroblasts and epithelial cells, it was found (see, Example 1) that when immersed in medium with viscosity comparable to that of the CF / IPF mucus, fibroblasts migrated 2-fold faster but epithelial cells moved 5-fold slower towards the wound. Subsequently, excessive fibroblasts at the wound site and slow re- epithelization cause scar tissue formation. Therefore, in addition to the known harmful effects such as airway obstruction, infection, and inflammation, highly viscous mucus may directly contribute to fibrosis in lung diseases and declined wound healing efficiency in older individuals. Leveraging these findings that cells with actively ruffling lamellapodia (e.g. fibroblasts, neutrophils, macrophages) accelerate and cells without decelerate (e.g. epithelial cells) at high viscosity, the inventors are developing drugs that transiently inducing lamellapodium in epithelial cells and / or specifically inhibiting fibroblast / immune cell motility can mitigate fibrogenesis and improve wound healing. These drugs either induce transient epithelial- mesenchymal transition (EMT) in epithelial cells, or inhibit fibroblast and immune cell migration, and delivery of these drugs is accomplished via mucus-penetrating nanoparticles.

[0055] Delivery Vehicles: Mucus Penetrating Peptide (MPP)-Decorated Particles.

[0056] Mucus is an elastic viscous substance composed of carbohydrates, protein, lipids, antibody, cellular debris, including DNA and RNA released from dead cells,bacteria and inorganic salts. The barrier properties of mucus are rooted in its dense network of mucin fibers, which contain highly glycosylated (negatively charged) segment, thus show high affinity with positively charged particles. For example, the diffusion rate of neutral polyacrylic acid (PAA)- polypropylene amide (PAM) nanoparticles (NPs) is 2.5-fold higher than positively charged PAM nanoparticles (NPs) (F. Laffleur, F. Hintzen, G. Shahnaz, et al. Nanomedicine (Lond), 9 (2014), pp.387-396). Similar phenomenon also has been reported by other groups that NPs with positively charged surface can be trapped in mucus effectively own to strong electrostatic interactions.

[0057] Additionally, there exist periodic hydrophobic domains along the mucin strains, which can bind hydrophobic particles with high avidity. Although hydrophobic interactions effectively limit the transport of some harmful agents such as bacteria, it also represents a challenge for the delivery of drug carriers, since the commonly used biomaterials are hydrophobic, like poly (lactic-co-glycolic acid) (PLGA) and polystyrene (PS). However, after coating PLGA NPs with hydrophilic molecules such as DNA, the average transport rates can be improved 10-fold in reconstituted pig gastric mucus.

[0058] Delivery vehicles for therapy comprising a cargo and having mucus-penetrating as well as cell-penetrating properties. Delivery of therapeutics, such as nucleic acids, small molecules, biologics and large molecules to mucosal epithelial cells for therapeutic purposes is made challenging by the physical barrier of the mucus. Accordingly, provided herein are delivery vehicles to penetrate a mucus layer and carry a cargo to the target tissues and cells. The delivery vehicles herein include mucus-penetrating features such as mucus-penetrating delivery vehicle compositions and mucus-penetrating polymer coatings and they are further coupled with mucus-penetrating peptides (MPPs) to have increased transport ability through the mucus associated with the target tissues.

[0059] The delivery vehicle may be a nanoparticle. In some cases, a delivery vehicle can have a diameter of from about 10 nm to about 100 nm, from about 100 nm to about 200 nm, from about 200 nm to about 300 nm, from about 300 nm to about 400 nm, and from about 400 nm to about 500 nm as measured by dynamic light scattering. The nanoparticle delivery vehicle may have a diameter of no more than 500 nm, no more than about 200 nm or no more than about100 nm. In some embodiments, a delivery vehicle can be from about 1 nm to about 150 nm in diameter. In some embodiments, the nanoparticle is a lipid-containing nanoparticle. In some cases, a delivery vehicle can include a lipid structure such as a lipid nanoparticle, a liposome, a liposomal polyplex, or a lipoplex.

[0060] The compositions provided herein include delivery vehicles, including nanoparticles, where the delivery vehicle itself has at least one mucus-penetrating feature. Such mucus-penetrating features include, for example, a zwitterionic feature of the delivery vehicle or a lipid composition that confers mucus-penetrating properties to the delivery vehicle. A zwitterionic feature may include the formation of a delivery vehicle such as a nanoparticle with chitosan / chitosanate or DLPC lipid nanoparticles.

[0061] The mucus-penetrating feature may be a mucus-penetrating surface modification of the delivery vehicle, such as a mucus-penetrating surface modification of a nanoparticle. The surface modification may be one or more of polyethylene glycol, poly (2-alkyl-2-oxazoline), poly(2-ethyl-2-oxazoline), poly(2-n-propyl-2-oxazoline), and poly(2-methyl-2-oxazaline), a salt thereof, a di block polymer and a tri block polymer thereof. In some embodiments, the polyethylene glycol surface modification has an average molecular weight ranging from about 2000 Da to about 3000 Da.

[0062] In some embodiments, a delivery vehicle includes more than one mucus- penetrating feature selected from a zwitterionic feature, a mucus-penetrating lipid composition that confers properties to the delivery vehicle and a mucus-penetrating surface modification, and combinations thereof.

[0063] The compositions herein include those where the MPP is conjugated directly to the delivery vehicle. In other embodiments, the MPP is indirectly conjugated to the delivery vehicle. The MPP may be conjugated directly to the surface modification, covalently or non- covalently.

[0064] The MPPs for use in the compositions and with the methods provided herein are cell-internalizable peptides (CPPs) that additionally have at least one mucus-penetrating feature. The vast majority of CPPs are not MPPs. CPPs generally fall into three group of peptides: cationic, amphipathic and hydrophobic. Due to the physical properties of the mucus, these CPPs will adhere to the mucus.

[0065] The MPPs for use in the compositions and with the methods provided herein have characteristics that confer mucus-penetrating properties. In some embodiments, the MPPs herein have an average hydropathy of an amino acid sequence of the MPP as measured by a Hodges score of less than or equal to 10 at pH 7.

[0066] In certain embodiments, the MPP-decorated particle is a mucus penetrating liposome (MPL). Liposomes are used as carriers for drugs and antigens because they can serve several different purposes (Storm & Crommelin, Pharmaceutical Science & Technology Today, 1, 19-311998). Liposome encapsulated drugs are inaccessible to metabolizing enzymes. Conversely, body components (such as erythrocytes or tissues at the injection site) are not directly exposed to the full dose of the drug. The duration of drug action can be prolonged by liposomes because of a slower release of the drug in the body. Targeted liposomes change the distribution of the drug over the body. Cells use endocytosis or phagocytosis mechanisms to take up liposomes into the cytosol. Furthermore, liposomes can protect a drug against degradation (e.g. metabolic degradation). Although sometimes successful, liposomes have limitations. Liposomes not only deliver drugs to diseased tissue, but also rapidly enter the liver, spleen, kidneys and Reticuloendothelial Systems, and leak drugs while in circulation (Harris & Chess, Nature, March 2003, 2, 214-221).

[0067] Liposomes are biocompatible and biodegradable. They consist of an aqueous core entrapped by one or more bilayers composed of natural or synthetic lipids. Liposomes composed of natural phospholipids are biologically inert and weakly immunogenic, and they possess low intrinsic toxicity. Further, drugs with different lipophilicities can be encapsulated into liposomes: strongly lipophilic drugs are entrapped almost completely in the lipid bilayer, strongly hydrophilic drugs are located exclusively in the aqueous compartment, and drugs with intermediate log P easily partition between the lipid and aqueous phases, both in the bilayer and in the aqueous core. Liposomes can be classified according to their lamellarity (uni-, oligo-, and multi-lamellar vesicles), size (small, intermediate, or large) and preparation method (such as reverse phase evaporation vesicles, VETs). Unilamellar vesicles comprise one lipid bilayer and generally have diameters of 50-250 nm. They contain a large aqueous core and are preferentially used to encapsulate water-soluble drugs. Multilamellar vesicles comprise several concentric lipid bilayers in an onion-skin arrangement and have diameters of 1-5 μm. The high lipid content allows these multilamellar vesicles to passively entrap lipid-soluble drugs. Unilamellar vesiclesare described herein due to the need for a small diameter of less than one micron, more preferably less than 500 nm.

[0068] Selection of the appropriate lipids for liposome composition is governed by the factors of: (1) liposome stability, (2) phase transition temperature, (3) charge, (4) non-toxicity to mammalian systems, (5) encapsulation efficiency, (6) lipid mixture characteristics. The vesicle- forming lipids preferably have two hydrocarbon chains, typically acyl chains, and a head group, either polar or nonpolar. The hydrocarbon chains may be saturated or have varying degrees of unsaturation. There are a variety of synthetic vesicle-forming lipids and naturally-occurring vesicle-forming lipids, including the sphingolipids, ether lipids, sterols, phospholipids, particularly the phosphoglycerides, and the glycolipids, such as the cerebrosides and gangliosides.

[0069] Phosphoglycerides include phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, phosphatidylserine phosphatidylglycerol and diphosphatidylglycerol (cardiolipin), where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation. As used herein, the abbreviation “PC” stands for phosphatidylcholine, and “PS” stand for phosphatidylserine. Lipids containing either saturated and unsaturated fatty acids are widely available to those of skill in the art. Additionally, the two hydrocarbon chains of the lipid may be symmetrical or asymmetrical. The above-described lipids and phospholipids whose acyl chains have varying lengths and degrees of saturation can be obtained commercially or prepared according to published methods.

[0070] Exemplary phosphatidylcholines include dilauroyl phophatidylcholine, dimyristoylphophatidylcholine, dipalmitoylphophatidylcholine, distearoylphophatidyl-choline, diarachidoylphophatidylcholine, dioleoylphophatidylcholine, dilinoleoyl-phophatidylcholine, dierucoylphophatidylcholine, palmitoyl-oleoyl-phophatidylcholine, egg phosphatidylcholine, myristoyl-palmitoylphosphatidylcholine, palmitoyl-myristoyl-phosphatidylcholine, myristoyl- stearoylphosphatidylcholine, palmitoyl-stearoyl-phosphatidylcholine, stearoyl- palmitoylphosphatidylcholine, stearoyl-oleoyl-phosphatidylcholine, stearoyl- linoleoylphosphatidylcholine and palmitoyl-linoleoyl-phosphatidylcholine. Assymetric phosphatidylcholines are referred to as 1-acyl, 2-acyl-sn-glycero-3-phosphocholines, wherein the acyl groups are different from each other. Symmetric phosphatidylcholines are referred to as 1,2-diacyl-sn-glycero-3-phosphocholines. As used herein, the abbreviation “PC” refers to phosphatidylcholine. The phosphatidylcholine 1,2-dimyristoyl-sn-glycero-3-phosphocholine is abbreviated herein as “DMPC.” The phosphatidylcholine 1,2-dioleoyl-sn-glycero-3- phosphocholine is abbreviated herein as “DOPC.” The phosphatidylcholine 1,2-dipalmitoyl-sn- glycero-3-phosphocholine is abbreviated herein as “DPPC.”

[0071] In general, saturated acyl groups found in various lipids include groups having the trivial names propionyl, butanoyl, pentanoyl, caproyl, heptanoyl, capryloyl, nonanoyl, capryl, undecanoyl, lauroyl, tridecanoyl, myristoyl, pentadecanoyl, palmitoyl, phytanoyl, heptadecanoyl, stearoyl, nonadecanoyl, arachidoyl, heneicosanoyl, behenoyl, trucisanoyl and lignoceroyl. The corresponding IUPAC names for saturated acyl groups are trianoic, tetranoic, pentanoic, hexanoic, heptanoic, octanoic, nonanoic, decanoic, undecanoic, dodecanoic, tridecanoic, tetradecanoic, pentadecanoic, hexadecanoic, 3,7,11,15-tetramethylhexadecanoic, heptadecanoic, octadecanoic, nonadecanoic, eicosanoic, heneicosanoic, docosanoic, trocosanoic and tetracosanoic. Unsaturated acyl groups found in both symmetric and asymmetric phosphatidylcholines include myristoleoyl, palmitoleyl, oleoyl, elaidoyl, linoleoyl, linolenoyl, eicosenoyl and arachidonoyl. The corresponding IUPAC names for unsaturated acyl groups are 9-cis-tetradecanoic, 9-cis-hexadecanoic, 9-cis-octadecanoic, 9-trans-octadecanoic, 9-cis-12-cis- octadecadienoic, 9-cis-12-cis-15-cis-octadecatrienoic, 11-cis-eicosenoic and 5-cis-8-cis-11-cis- 14-cis-eicosatetraenoic.

[0072] Exemplary phosphatidylethanolamines include dimyristoyl- phosphatidylethanolamine, dipalmitoyl-phosphatidylethanolamine, distearoyl- phosphatidylethanolamine, dioleoyl-phosphatidylethanolamine and egg phosphatidylethanolamine. Phosphatidylethanolamines may also be referred to under IUPAC naming systems as 1,2-diacyl-sn-glycero-3-phosphoethanolamines or 1-acyl-2-acyl-sn-glycero- 3-phosphoethanolamine, depending on whether they are symmetric or assymetric lipids.

[0073] Exemplary phosphatidic acids include dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid and dioleoyl phosphatidic acid. Phosphatidic acids may also be referred to under IUPAC naming systems as 1,2-diacyl-sn-glycero-3-phosphate or 1-acyl-2-acyl-sn-glycero- 3-phosphate, depending on whether they are symmetric or assymetric lipids.

[0074] Exemplary phosphatidylserines include dimyristoyl phosphatidylserine, dipalmitoyl phosphatidylserine, dioleoylphosphatidylserine, distearoyl phosphatidylserine,palmitoyl-oleylphosphatidylserine and brain phosphatidylserine. Phosphatidylserines may also be referred to under IUPAC naming systems as 1,2-diacyl-sn-glycero-3-[phospho-L-serine] or 1- acyl-2-acyl-sn-glycero-3-[phospho-L-serine], depending on whether they are symmetric or assymetric lipids. As used herein, the abbreviation “PS” refers to phosphatidylserine.

[0075] Exemplary phosphatidylglycerols include dilauryloylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoyl-phosphatidylglycerol, dimyristoylphosphatidylglycerol, palmitoyl-oleoyl-phosphatidylglycerol and egg phosphatidylglycerol. Phosphatidylglycerols may also be referred to under IUPAC naming systems as 1,2-diacyl-sn-glycero-3-[phospho-rac-(1-glycerol)] or 1-acyl-2-acyl-sn-glycero-3- [phospho-rac-(1-glycerol)], depending on whether they are symmetric or assymetric lipids. The phosphatidylglycerol 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] is abbreviated herein as “DMPG”. The phosphatidylglycerol 1,2-dipalmitoyl-sn-glycero-3-(phospho-rac-1- glycerol) (sodium salt) is abbreviated herein as “DPPG”.

[0076] Suitable sphingomyelins might include brain sphingomyelin, egg sphingomyelin, dipalmitoyl sphingomyelin, and distearoyl sphingomyelin.

[0077] Other suitable lipids include glycolipids, sphingolipids, ether lipids, glycolipids such as the cerebrosides and gangliosides, and sterols, such as cholesterol or ergosterol. As used herein, the term cholesterol is sometimes abbreviated as “Chol.” Additional lipids suitable for use in liposomes are known to persons of skill in the art and are cited in a variety of sources, such as 1998 McCutcheon's Detergents and Emulsifiers, 1998 McCutcheon's Functional Materials, both published by McCutcheon Publishing Co., New Jersey, and the Avanti Polar Lipids, Inc. Catalog.

[0078] The overall surface charge of the liposome can affect the tissue uptake of a liposome. In certain embodiments of the present invention anionic phospholipids such as phosphatidylserine, phosphatidylinositol, phosphatidic acid, and cardiolipin are used. Neutral lipids such as dioleoylphosphatidyl ethanolamine (DOPE) may be used to target uptake of liposomes by specific tissues or to increase circulation times of intravenously administered liposomes. Cationic lipids may be used for alteration of liposomal charge, where the cationic lipid can be included as a minor component of the lipid composition or as a major or sole component. Suitable cationic lipids typically have a lipophilic moiety, such as a sterol, an acyl ordiacyl chain, and where the lipid has an overall net positive charge. Preferably, the head group of the lipid carries the positive charge.

[0079] The fluidity or rigidity of the liposome can be used to control factors such as the stability of the liposome in serum or the rate of release of the entrapped agent in the liposome. Liposomes having a more rigid lipid bilayer, or a liquid crystalline bilayer, are achieved by incorporation of a relatively rigid lipid. The rigidity of the lipid bilayer correlates with the phase transition temperature of the lipids present in the bilayer. Phase transition temperature is the temperature at which the lipid changes physical state and shifts from an ordered gel phase to a disordered liquid crystalline phase. Several factors affect the phase transition temperature of a lipid including hydrocarbon chain length and degree of unsaturation, charge and headgroup species of the lipid. Lipid having a relatively high phase transition temperature will produce a more rigid bilayer. Other lipid components, such as cholesterol, are also known to contribute to membrane rigidity in lipid bilayer structures. Cholesterol is widely used by those of skill in the art to manipulate the fluidity, elasticity and permeability of the lipid bilayer. It is thought to function by filling in gaps in the lipid bilayer. In contrast, lipid fluidity is achieved by incorporation of a relatively fluid lipid, typically one having a lower phase transition temperature. Phase transition temperatures of many lipids are tabulated in a variety of sources, such as Avanti Polar Lipids catalogue and Lipidat by Martin Caffrey, CRC Press.

[0080] Liposomes are preferably made from endogenous phospholipids such as dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG), phosphatidyl serine, phosphatidyl choline, dioleoyphosphatidyl choline [DOPC], cholesterol (CHOL) and cardiolipin.

[0081] Among the different polymers investigated in attempts to improve the blood circulation time of liposomes, poly-(ethylene glycol) (PEG) has been widely used as polymeric steric stabilizer. It can be incorporated on the liposomal surface in different ways, but the most widely used method at present is to anchor the polymer in the liposomal membrane via a cross- linked lipid (ie, PEG-distearoylphosphatidylethanolamine [DSPE]. PEG (CAS number 25322- 68-3) is a linear polyether diol with many useful properties, such as biocompatibility (Powell G M. Polyethylene glycol. In: Davidson R L, editor. Handbook of water soluble gums and resins. McGraw-Hill: 1980. pp.18-31), solubility in aqueous and organic media, lack of toxicity, very low immunogenicity and antigenicity (Dreborg et al. Crit Rev Ther Drug Carrier Syst.1990:315-65), and good excretion kinetics (Yamaoka et al. J Pharm Sci.1994; 83:601-6). The molecular weight and structure of PEG molecules can be freely modulated for specific purposes, and it is easier and cheaper to conjugate the polymer with the lipid.

[0082] Poly-ethylene glycols have been used to derivatize therapeutic proteins and peptides, increasing drug stability and solubility, lowering toxicity, increasing half-life (Caliceti et al. Adv Drug Del Rev.2003; 55:1261-77), decreasing clearance and immunogenicity. These benefits have been particularly observed using branched PEG in the derivatization (Monfardini et al. Bioconj Chem.1998; 9:418-50). For the most part, reaction with PEG derivatives does not alter the mechanism of action of a therapeutic protein; rather it enhances its therapeutic effect by altering its pharmacokinetics. PEG-ademase (utilized to treat immunodeficiency), PEG-visomant (human growth hormone), PEG-aspargase (for leukemias), PEG-interferon-alpha (for chronic hepatitis C), PEG-aldesleukin (PEG-IL-2) (an anticancer agent), and PEG-filgrastim (for chemotherapy-induced transferase neutropenia) are the principal PEGylated proteins in clinical use (Mahmood et al. Clin Pharmacokinet.2005; 44:331-47).

[0083] Surface modification of liposomes with PEG can be achieved in several ways: by physically adsorbing the polymer onto the surface of the vesicles, by incorporating the PEG-lipid conjugate during liposome preparation, or by covalently attaching reactive groups onto the surface of preformed liposomes. Grafting PEG onto liposomes has demonstrated several biological and technological advantages. The most significant properties of PEGylated vesicles are their strongly reduced MPS uptake and their prolonged blood circulation and thus improved distribution in perfused tissues. Moreover, the PEG chains on the liposome surface avoid vesicle aggregation, improving stability of formulations.

[0084] Liposomes, coated with one or more materials that promote diffusion of the particles through mucosa are disclosed. Examples of the surface-altering agents include, but are not limited to, polyethylene glycol (“PEG”) and poloxomers (polyethylene oxide block copolymers). Poly(ethylene glycol) (PEG) are macromolecules which can be used for modification of biological macromolecules and many pharmaceutical and biotechnological applications. Liposomes can be modified by combining them with PEG.

[0085] Polyethylene Glycol (PEG): A coating agent is poly(ethylene glycol), also known as PEG. PEG may be employed to reduce adhesion in brain ECM in certain configurations, e.g., wherein the length of PEG chains extending from the surface is controlled (such that long,unbranched chains that interpenetrate into the ECM are reduced or eliminated). For example, linear high MW PEG may be employed in the preparation of particles such that only portions of the linear strands extend from the surface of the particles (e.g., portions equivalent in length to lower MW PEG molecules). Alternatively, branched high MW PEG may be employed. In such embodiments, although the molecular weight of a PEG molecule may be high, the linear length of any individual strand of the molecule that extends from the surface of a particle would correspond to a linear chain of a lower MW PEG molecule.

[0086] Representative PEG molecular weights in daltons (Da) include up to or at least 300 Da, 600 Da, 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 8 kDa, 10 kDa, 15 kDa, 20 kDa, 30 kDa, 50 kDa, 100 kDa, 200 kDa, 500 kDa, and 1 MDa. In preferred embodiments, the PEG has a molecular weight of about 2,000 to 5,000 Daltons. PEG of any given molecular weight may vary in other characteristics such as length, density, and branching. In a particular embodiment, a coating agent is methoxy-PEG-amine, with a MW of 5 kDa. In another embodiment, a coating agent is methoxy-PEG-N-hydroxysuccinimide with a MW of 5 kDa (mPEG-NHS 5 kDa).

[0087] In alternative embodiments, the coating is a poloxamer such as the polyethylene glycol-polyethylene oxide block copolymers marketed as PLUORONICs®.

[0088] PEG alternative polymers should be soluble, hydrophilic, have highly flexible main chain, and high biocompatibility. Synthetic polymers, such as poly(vinyl pyrrolidone) (PVP) and poly(acryl amide) (PAA), are the most prominent examples of other potentially protective polymers (Torchilin et al Biochim Biophys Acta.1994 Oct.12; 1195(1):181-4; Biochim Biophys Acta.1994 Oct.12; 1195(1):11-20; J Pharm Sci.1995 September; 84(9):1049- 53). Liposomes containing DSPE covalently linked to poly(2-methyl-2-oxazoline) or to poly(2- ethyl-2-oxazoline) also exhibit extended blood circulation time and decreased uptake by the liver and spleen (Woodle, et al. Bioconjug Chem.1994 November-December; 5(6):493-6). Similar observations have been reported for phosphatidyl polyglycerols (Unezaki, et al. Pharm Res.1994 August; 11(8):1180-5).

[0089] Long circulating liposomes are prepared using poly[N-(2-hydroxypropyl) methacrylamide] (Whiteman, et al. J Liposome Res.2001; 11(2-3):153-64), amphiphilic poly-N- vinylpyrrolidones (Torchilin Biomaterials.2001 November; 22(22):3035-44.), L-amino-acid- based biodegradable polymer-lipid conjugates (Metselaar, et al. Bioconjug Chem.2003 November-December; 14(6):1156-64), and polyvinyl alcohol (Takeuchi, et al. Eur. J. Pharm.Biopharm, 2012 February; 80(2):340-6. doi:10.1016 / j.ejpb.2011.10.011. Epub 2011 Oct.20.). All groups of polymer-coated liposomes reported have been found to extend blood circulation time, while liver capture was diminished. These results are comparable with those for PEG- liposomes; the efficacy of the steric effect quite naturally depends on the quantity of incorporated polymer. The prolonged circulation time of polyvinyl alcohol-(molecular weight: 20000) coated liposomes (1.3 mol % coating) was comparable with that of a stealth liposome prepared with 8 mol % of DSPE-PEG2000.

[0090] Also, L-amino-acid-based polymers also showed prolonged circulation time and reduced uptake by the MPS, to the same extent as DSPE-PEG2000. Furthermore, these polymers appear to be attractive alternatives for designing long-circulation liposomes, because they have the advantage of being biodegradable.

[0091] PEG-coated liposomes have also been shown to increase mucosal penetration. See, for example, Li, et al. Int. J. Nanomed.2011: 6,3151-3162 and WO2013166498 by The Johns Hopkins University. It should be noted that the formulations described herein represent a subset with improved mucosal penetration as compared to PEG-coated liposomes generally, as demonstrated by the examples, showing that there is a narrow range of the ratio of PEG-lipid to lipid mol % to provide optimized mucosal penetration.

[0092] Additional materials which can be used in the present composition including MPLs are disclosed in U.S. Patent 10,566,017 to Hanes et al.; US 2023 / 0108636 to Hanes t al.; and PCT / US2011 / 059321, all incorporated herein by reference in their entirety..

[0093] Targeting moieties: In some aspects, MPLs disclosed can further comprise a targeting moiety, in addition to a therapeutic agent. The targeting moiety can be a biological molecule, such as an antibody or antigen binding fragments thereof, aptamer, a protein, a peptide, a lipid, or a carbohydrate, or a synthetic molecule. For example, the targeting moiety can be an affinity ligand (e.g., antibody, VHH domain, phage display peptide, fibronectin domain, camelid, aptamer, VNAR), a synthetic polymer (e.g., PEG), a natural ligand / molecule (e.g., CD40L, albumin ,CD47, CD24, CD55, CD59), a recombinant protein, but not limited thereto.

[0094] In certain aspects, the targeting moiety, is displayed on the surface of MPLs. The targeting moiety can be displayed on the MPL surface by being fused to a scaffold protein (e.g., as a genetically encoded fusion molecule). In some aspects, the targeting moiety can bedisplayed on the MPL surface by chemical reaction attaching targeting moiety to an MPL surface molecule. A non-limiting example is PEGylation.

[0095] In certain embodiments, an MPL, comprises an extracellular vesicle isolated from a biological cell with at least one lipid incorporated into the membrane thereof.

[0096] In this embodiment the lipid may be a synthetic lipid or an exogenous lipid / non- native lipid or a combination thereof. A synthetic lipid comprises 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), dipalmitoylphosphatidylcholine (DPPC), or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DOTAP) or a combination thereof. Also, the biological cell may be associated with a pathophysiological condition. In one aspect the pathophysiological condition may be a cancer. In another aspect the pathophysiological condition is cystic fibrosis, COPD, asthma, tuberculosis, or an infection, such as by a virus, bacteria or parasite. In another aspect the biological cell may be a primary mesenchymal stem cell, an embryonic kidney cell, an embryonic fibroblast cell, an alveolar basal epithelial cell, or a monocytic cell or an immortalized cell-line thereof.

[0097] Density of Coating Agent: In certain embodiments the liposomes are coated with PEG or other coating agents at a density that optimizes rapid diffusion through the brain parenchyma. The density of the coating can be varied based on a variety of factors including the material and the composition of the particle.

[0098] For liposomes, the composition is usually defined by the molar ratio between PEG-lipid and non-PEGylated-lipid. These can range from three to eleven mol %. Most preferably the ratio of PEG-lipid to non-PEGylated-lipid is about 7 mol %.

[0099] Liposome Formation and Drug Entrapment: The formation and use of liposomes is generally known to those of skill in the art, as described in, e.g. Liposome Technology, Vols. 1, 2 and 3, Gregory Gregoriadis, ed., CRC Press, Inc; Liposomes: Rational Design, Andrew S. Janoff, ed., Marcel Dekker, Inc.; Medical Applications of Liposomes, D. D. Lasic and D. Papahadjopoulos, eds., Elsevier Press; Bioconjugate Techniques, by Greg T. Hermanson, Academic Press; and Pharmaceutical Manufacturing of Liposomes, by Francis J. Martin, in Specialized Drug Delivery Systems (Praveen Tyle, Ed.), Marcel Dekker, Inc. [000100] The original method of forming liposomes (Bangham et al., 1965, J. Mol. Biol. 13: 238-252) involved first suspending phospholipids in an organic solvent and then evaporating to dryness until a dry lipid cake or film is formed. An appropriate amount of aqueous medium isadded and the lipids spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). These MLVs can then be dispersed by mechanical means. [000101] While the original MLVs and SUVs were created using phospholipids, any of the lipid compositions described previously can be used to create MLVs and SUVs. When mixtures of lipids are used the lipids are typically co-dissolved in an organic solvent prior to the evaporation step of the process described above. [000102] An alternate method of creating large unilamellar vesicles (LUVs) is the reverse- phase evaporation process, described, for example, in U.S. Pat. No.4,235,871. This process generates reverse-phase evaporation vesicles (REVs), which are mostly unilamellar but also typically contain some oligolamellar vesicles. In this procedure a mixture of polar lipid in an organic solvent is mixed with a suitable aqueous medium. A homogeneous water-in-oil type of emulsion is formed and the organic solvent is evaporated until a gel is formed. The gel is then converted to a suspension by dispersing the gel-like mixture in an aqueous media. [000103] Liposomes may also be prepared wherein the liposomes have substantially homogeneous sizes in a selected size range. One effective sizing method for REVs and MLVs involves extruding an aqueous suspension of the liposomes through a series of polycarbonate membranes having a selected uniform pore size in the range of 0.03 to 0.2 micron, typically 0.05, 0.08, 0.1, or 0.2 microns. The pore size of the membrane corresponds roughly to the largest sizes of liposomes produced by extrusion through that membrane, particularly where the preparation is extruded two or more times through the same membrane. Homogenization methods are also useful for down-sizing liposomes to sizes of 100 nm or less (Martin, F. J., in Specialized Drug Delivery Systems-Manufacturing and Production Technology, (P. Tyle, Ed.) Marcel Dekker, New York, pp.267-316 (1990)). Homogenization relies on shearing energy to fragment large liposomes into smaller ones. Other appropriate methods of down-sizing liposomes include reducing liposome size by vigorous agitation of the liposomes in the presence of an appropriate solubilizing detergent, such as deoxycholate. [000104] Therapeutic and Prophylactic Agents: In some embodiments, the particles have encapsulated therein, dispersed therein, and / or covalently or non-covalently associate 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.[000105] Any protein can be formulated, including recombinant, isolated, or synthetic proteins, glycoproteins, or lipoproteins. These may be antibodies (including antibody fragments and recombinant antibodies), enzymes, growth factors or hormones, immunomodulators, antisense reagents, siRNA reagents, peptides, organic or inorganic molecules, natural or synthetic compounds, antiinfectives, antiproliferatives, or other therapeutic, prophylactic, or diagnostic proteins. In certain embodiments, the protein has a molecular weight greater than about 150 kDa, greater than 160 kDa, greater than 170 kDa, greater than 180 kDa, greater than 190 kDa or even greater than 200 kDa. In certain embodiments, the protein can be a PEGylated protein. [000106] 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. [000107] Pharmaceutical Formulations [000108] The formulations described herein contain an effective amount of liposomes 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, vaginally, rectally, or orally), or via pulmonary administration. [000109] Pulmonary Formulations: Pharmaceutical formulations and methods for the pulmonary administration of active agents to patients are known in the art. [000110] 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 bronchioli. The upper and lower airways are called the conducting airways. The terminal bronchioli then divide into respiratory bronchioli whichthen lead to the ultimate respiratory zone, the alveoli, or deep lung, where the exchange of gases occurs. [000111] 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. [000112] Dry powder formulations are finely divided solid formulations containing liposome carriers which are suitable for pulmonary administration. Dry powder formulations include, at a minimum, one or more liposome carriers 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. [000113] In other embodiments, the dry powder formulations contain one or more liposome gene carriers in combination with a pharmaceutically acceptable carrier. In these embodiments, the liposome gene carriers and pharmaceutical carrier can be formed into nano- or microparticles for delivery to the lung. [000114] The pharmaceutical carrier may include a bulking agent or a lipid or surfactant. Natural surfactants such as dipalmitoylphosphatidylcholine (DPPC) are the most preferred. Synthetic and animal derived pulmonary surfactants include: [000115] Synthetic Pulmonary Surfactants: 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. Venticute--DPPC, PG, palmitic acid and recombinant SP-C. [000116] Animal Derived Surfactants: Alveofact—extracted from cow lung lavage fluid. Curosurf—extracted from material derived from minced pig lung. Infasurf—extracted from calf lung lavage fluid. Survanta—extracted from minced cow lung with additional DPPC, palmitic acid and tripalmitin. Exosurf, Curosurf, Infasurf, and Survanta are the surfactants currently FDA approved for use in the U.S. [000117] The pharmaceutical carrier may also include one or more stabilizing agents or dispersing agents. The pharmaceutical carrier may also include one or more pH adjusters orbuffers. 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. [000118] Dry powder formulations are typically prepared by blending one or more liposome carriers with one or more pharmaceutically 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., air-attrition milling (jet milling), ball milling), high pressure homogenization, and / or supercritical fluid crystallization. [000119] 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. [000120] 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. [000121] Liquid formulations contain one or more liposome carriers suspended in a liquid pharmaceutical carrier. [000122] 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. [000123] Preferably, liquid formulations are isotonic relative to physiological fluids and of approximately the same pH, ranging e.g., from about pH 4.0 to about pH 7.4, more preferably from about pH 6.0 to pH 7.0. The liquid pharmaceutical carrier can include one or morephysiologically 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. [000124] 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- hydroxybenzoate. [000125] 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, tetrahydofuran, 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. [000126] 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. [000127] 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. [000128] 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. [000129] 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.[000130] Parenteral and Enteral Formulations: In some embodiments, the liposomes are formulated for parenteral delivery, such as injection or infusion, in the faun 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. In some embodiments, the liposomes are formulated for parenteral formulation to the eye. [000131] “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. [000132] 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. [000133] 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, corn 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 many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. [000134] 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. [000135] 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, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-beta-alanine, sodium N-lauryl-beta-iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine. [000136] 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). [000137] 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. [000138] 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. [000139] 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, the preferred methods of preparation are vacuum-drying and freeze-drying techniqueswhich 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. [000140] Ocular Formulations: Pharmaceutical formulations for ocular administration are preferably in the form of a sterile aqueous solution or suspension of particles formed from one or more polymer-drug conjugates. Acceptable solvents include, for example, water, Ringer's solution, phosphate buffered saline (PBS), and isotonic sodium chloride solution. The formulation may also be a sterile solution, suspension, or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as 1,3-butanediol. [000141] In some instances, the formulation is distributed or packaged in a liquid form. Alternatively, formulations for ocular administration can be packed as a solid, obtained, for example by lyophilization of a suitable liquid formulation. The solid can be reconstituted with an appropriate carrier or diluent prior to administration. [000142] Solutions, suspensions, or emulsions for ocular administration may be buffered with an effective amount of buffer necessary to maintain a pH suitable for ocular administration. Suitable buffers are well known by those skilled in the art and some examples of useful buffers are acetate, borate, carbonate, citrate, and phosphate buffers. [000143] Solutions, suspensions, or emulsions for ocular administration may also contain one or more tonicity agents to adjust the isotonic range of the formulation. Suitable tonicity agents are well known in the art and some examples include glycerin, mannitol, sorbitol, sodium chloride, and other electrolytes. [000144] Solutions, suspensions, or emulsions for ocular administration may also contain one or more preservatives to prevent bacterial contamination of the ophthalmic preparations. Suitable preservatives are known in the art, and include polyhexamethylenebiguanidine (PHMB), benzalkonium chloride (BAK), stabilized oxychloro complexes (otherwise known as Purite®), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, thimerosal, and mixtures thereof. [000145] Solutions, suspensions, or emulsions for ocular administration may also contain one or more excipients known art, such as dispersing agents, wetting agents, and suspending agents.[000146] Topical Formulations: [000147] In still other embodiments, the liposomes 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. [000148] In some embodiments, the liposomes can be administered as a liquid formulation, such as a solution or suspension, a semi-solid formulation, such as an lotion or ointment, or a solid formulation. In some embodiments, the liposomes 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. [000149] The formulation may contain one or more excipients, such as emollients, surfactants, emulsifiers, and penetration enhancers. [000150] “Emollients” are an externally applied agent that softens or soothes skin and are generally known in the art and listed in compendia, such as the “Handbook of Pharmaceutical Excipients”, 4th Ed., Pharmaceutical Press, 2003. These include, without limitation, almond oil, castor oil, ceratonia extract, cetostearoyl alcohol, cetyl alcohol, cetyl esters wax, cholesterol, cottonseed oil, cyclomethicone, ethylene glycol palmitostearate, glycerin, glycerin monostearate, glyceryl monooleate, isopropyl myristate, isopropyl palmitate, lanolin, lecithin, light mineral oil, medium-chain triglycerides, mineral oil and lanolin alcohols, petrolatum, petrolatum and lanolin alcohols, soybean oil, starch, stearyl alcohol, sunflower oil, xylitol and combinations thereof. In one embodiment, the emollients are ethylhexylstearate and ethylhexyl palmitate. [000151] “Surfactants” are surface-active agents that lower surface tension and thereby increase the emulsifying, foaming, dispersing, spreading and wetting properties of a product. Suitable non-ionic surfactants include emulsifying wax, glyceryl monooleate, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polysorbate, sorbitan esters, benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin monostearate, poloxamer, povidone and combinations thereof. In one embodiment, the non-ionic surfactant is stearyl alcohol.[000152] “Emulsifiers” are surface active substances which promote the suspension of one liquid in another and promote the formation of a stable mixture, or emulsion, of oil and water. Common emulsifiers are: metallic soaps, certain animal and vegetable oils, and various polar compounds. Suitable emulsifiers include acacia, anionic emulsifying wax, calcium stearate, carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol, diethanolamine, ethylene glycol palmitostearate, glycerin monostearate, glyceryl monooleate, hydroxpropyl cellulose, hypromellose, lanolin, hydrous, lanolin alcohols, lecithin, medium-chain triglycerides, methylcellulose, mineral oil and lanolin alcohols, monobasic sodium phosphate, monoethanolamine, nonionic emulsifying wax, oleic acid, poloxamer, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, propylene glycol alginate, self-emulsifying glyceryl monostearate, sodium citrate dehydrate, sodium lauryl sulfate, sorbitan esters, stearic acid, sunflower oil, tragacanth, triethanolamine, xanthan gum and combinations thereof. In one embodiment, the emulsifier is glycerol stearate. [000153] Suitable classes of penetration enhancers are known in the art and include, but are not limited to, fatty alcohols, fatty acid esters, fatty acids, fatty alcohol ethers, amino acids, phospholipids, lecithins, cholate salts, enzymes, amines and amides, complexing agents (liposomes, cyclodextrins, modified celluloses, and diimides), macrocyclics, such as macrocylic lactones, ketones, and anhydrides and cyclic ureas, surfactants, N-methyl pyrrolidones and derivatives thereof, DMSO and related compounds, ionic compounds, azone and related compounds, and solvents, such as alcohols, ketones, amides, polyols (e.g., glycols). Examples of these classes are known in the art. [000154] “Hydrophilic” as used herein refers to substances that have strongly polar groups that readily interact with water. [000155] “Lipophilic” refers to compounds having an affinity for lipids. [000156] “Amphiphilic” refers to a molecule combining hydrophilic and lipophilic (hydrophobic) properties [000157] “Hydrophobic” as used herein refers to substances that lack an affinity for water; tending to repel and not absorb water as well as not dissolve in or mix with water. [000158] A “gel” is a colloid in which the dispersed phase has combined with the continuous phase to produce a semisolid material, such as jelly.[000159] An “oil” is a composition containing at least 95% wt of a lipophilic substance. Examples of lipophilic substances include but are not limited to naturally occurring and synthetic oils, fats, fatty acids, lecithins, triglycerides and combinations thereof. [000160] A “continuous phase” refers to the liquid in which solids are suspended or droplets of another liquid are dispersed, and is sometimes called the external phase. This also refers to the fluid phase of a colloid within which solid or fluid particles are distributed. If the continuous phase is water (or another hydrophilic solvent), water-soluble or hydrophilic drugs will dissolve in the continuous phase (as opposed to being dispersed). In a multiphase formulation (e.g., an emulsion), the discreet phase is suspended or dispersed in the continuous phase. [000161] An “emulsion” is a composition containing a mixture of non-miscible components homogenously blended together. In particular embodiments, the non-miscible components include a lipophilic component and an aqueous component. An emulsion is a preparation of one liquid distributed in small globules throughout the body of a second liquid. The dispersed liquid is the discontinuous phase, and the dispersion medium is the continuous phase. When oil is the dispersed liquid and an aqueous solution is the continuous phase, it is known as an oil-in-water emulsion, whereas when water or aqueous solution is the dispersed phase and oil or oleaginous substance is the continuous phase, it is known as a water-in-oil emulsion. Either or both of the oil phase and the aqueous phase may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. Preferred excipients include surfactants, especially non- ionic surfactants; emulsifying agents, especially emulsifying waxes; and liquid non-volatile non- aqueous materials, particularly glycols such as propylene glycol. The oil phase may contain other oily pharmaceutically approved excipients. For example, materials such as hydroxylated castor oil or sesame oil may be used in the oil phase as surfactants or emulsifiers. [000162] A sub-set of emulsions are the self-emulsifying systems. These drug delivery systems are typically capsules (hard shell or soft shell) comprised of the drug dispersed or dissolved in a mixture of surfactant(s) and lipophilic liquids such as oils or other water immiscible liquids. When the capsule is exposed to an aqueous environment and the outer gelatin shell dissolves, contact between the aqueous medium and the capsule contents instantly generates very small emulsion droplets. These typically are in the size range of micelles orliposomes. No mixing force is required to generate the emulsion as is typically the case in emulsion formulation processes. [000163] A “lotion” is a low- to medium-viscosity liquid formulation. A lotion can contain finely powdered substances that are in soluble in the dispersion medium through the use of suspending agents and dispersing agents. Alternatively, lotions can have as the dispersed phase liquid substances that are immiscible with the vehicle and are usually dispersed by means of emulsifying agents or other suitable stabilizers. In one embodiment, the lotion is in the form of an emulsion having a viscosity of between 100 and 1000 centistokes. The fluidity of lotions permits rapid and uniform application over a wide surface area. Lotions are typically intended to dry on the skin leaving a thin coat of their medicinal components on the skin's surface. [000164] A “cream” is a viscous liquid or semi-solid emulsion of either the “oil-in-water” or “water-in-oil type”. Creams may contain emulsifying agents and / or other stabilizing agents. In one embodiment, the formulation is in the form of a cream having a viscosity of greater than 1000 centistokes, typically in the range of 20,000-50,000 centistokes. Creams are often time preferred over ointments as they are generally easier to spread and easier to remove. [000165] The difference between a cream and a lotion is the viscosity, which is dependent on the amount / use of various oils and the percentage of water used to prepare the formulations. Creams are typically thicker than lotions, may have various uses and often one uses more varied oils / butters, depending upon the desired effect upon the skin. In a cream formulation, the water- base percentage is about 60-75% and the oil-base is about 20-30% of the total, with the other percentages being the emulsifier agent, preservatives and additives for a total of 100%. [000166] An “ointment” is a semisolid preparation containing an ointment base and optionally one or more active agents. Examples of suitable ointment bases include hydrocarbon bases (e.g., petrolatum, white petrolatum, yellow ointment, and mineral oil); absorption bases (hydrophilic petrolatum, anhydrous lanolin, lanolin, and cold cream); water-removable bases (e.g., hydrophilic ointment), and water-soluble bases (e.g., polyethylene glycol ointments). Pastes typically differ from ointments in that they contain a larger percentage of solids. Pastes are typically more absorptive and less greasy that ointments prepared with the same components. [000167] A “gel” is a semisolid system containing dispersions of small or large molecules in a liquid vehicle that is rendered semisolid by the action of a thickening agent or polymeric material dissolved or suspended in the liquid vehicle. The liquid may include a lipophiliccomponent, an aqueous component or both. Some emulsions may be gels or otherwise include a gel component. Some gels, however, are not emulsions because they do not contain a homogenized blend of immiscible components. Suitable gelling agents include, but are not limited to, modified celluloses, such as hydroxypropyl cellulose and hydroxyethyl cellulose; Carbopol homopolymers and copolymers; and combinations thereof. Suitable solvents in the liquid vehicle include, but are not limited to, diglycol monoethyl ether; alklene glycols, such as propylene glycol; dimethyl isosorbide; alcohols, such as isopropyl alcohol and ethanol. The solvents are typically selected for their ability to dissolve the drug. Other additives, which improve the skin feel and / or emollience of the formulation, may also be incorporated. Examples of such additives include, but are not limited, isopropyl myristate, ethyl acetate, C12-C15 alkyl benzoates, mineral oil, squalane, cyclomethicone, capric / caprylic triglycerides, and combinations thereof. [000168] Foams consist of an emulsion in combination with a gaseous propellant. The gaseous propellant consists primarily of hydrofluoroalkanes (HFAs). Suitable propellants include HFAs such as 1,1,1,2-tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3-heptafluoropropane (HFA 227), but mixtures and admixtures of these and other HFAs that are currently approved or may become approved for medical use are suitable. The propellants preferably are not hydrocarbon propellant gases which can produce flammable or explosive vapors during spraying. Furthermore, the compositions preferably contain no volatile alcohols, which can produce flammable or explosive vapors during use. [000169] Buffers are used to control pH of a composition. Preferably, the buffers buffer the composition from a pH of about 4 to a pH of about 7.5, more preferably from a pH of about 4 to a pH of about 7, and most preferably from a pH of about 5 to a pH of about 7. In a preferred embodiment, the buffer is triethanolamine. [000170] Preservatives can be used to prevent the growth of fungi and microorganisms. Suitable antifungal and antimicrobial agents include, but are not limited to, benzoic acid, butylparaben, ethyl paraben, methyl paraben, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, and thimerosal. [000171] In certain embodiments, it may be desirable to provide continuous delivery of one or more noscapine analogs to a patient in need thereof. For topical applications, repeatedapplication can be done or a patch can be used to provide continuous administration of the noscapine analogs over an extended period of time. [000172] The formulations including drug-encapsulated mucus-penetrating liposomes suitably comprise one or more therapeutic agents. [000173] In one aspect, the formulations including drug-encapsulated mucus-penetrating liposomes suitably comprise one or more microtubule stabilizing drugs such as e.g. ixabepilone, cabazitaxel, nab-paclitaxel, docetaxel and / or nocodazole. [000174] In another aspect, the formulations including drug-encapsulated mucus-penetrating liposomes suitably comprise one or more LIMK / cofilin inhibitors such as e.g. LIMK1 inhibitor BMS-4, CRT0105446, CRT0105950; LIJTF500025 , LIMKi3, and / or FRAX486. [000175] In another aspect, the formulations including drug-encapsulated mucus-penetrating liposomes suitably comprise one or more Dia1 inhibitors such as SMIFH2. [000176] In another aspect, the formulations including drug-encapsulated mucus- penetrating liposomes suitably comprise one or more YAP inhibitors such as YAP / TAZ inhibitor-1, Trichostatin A, Verteporfin, quinolinols, and / or dihydrexidine, DHX. [000177] In another aspect, the formulations including drug-encapsulated mucus-penetrating liposomes suitably comprise one or more proliferation modulation agents such as Mitomycin C for fibroblasts and macrophages and / or IL1 for epithelial cells. [000178] In another aspect, the formulations including drug-encapsulated mucus- penetrating liposomes suitably comprise one or more FAK / Src inhibitors such as TAE226 (NVP- 226), VS-6063(PF-04554878), defactinib and / or dasatinib. [000179] In another aspect, the formulations including drug-encapsulated mucus-penetrating liposomes suitably comprise one or more Modulators of Ca 2+ ion channel activity such as e.g. Amlodipine (Norvasc®), Diltiazem (Cardiazem®, Tiazac®, Tiazac® XC ), Felodipine (Plendil®), Nifedipine XL (Adalat XL®), Verapamil, GsMTx4 (Piezo1 and Piezo2 inhibitor), GSK101 (TRPV4 agonist), Capsaicin (TRPV1 agonist), AZD-1386 (TRPV1 inhibitor), JNJ‐ 39439335 (Mavatrep), XEN-D0501 (TRPV1 inhibitor), GSK-2798745 (TRPV4 inhibitor), GSK- 2193874 (TRPV4 Inhibitor) and / or HC-067047 (TRPV4 Inhibitor). EXAMPLES [000180] EXAMPLE 1 MEMBRANE RUFFLING IS A MECHANOSENSOR OF EXTRACELLULAR FLUID VISCOSITY.[000181] The effects of ECF viscosity on cell behavior at the subcellular scale were investigated to gain mechanistic insight into how cells sense and respond to viscosity. Counterintuitively, it was found that cells in highly viscous medium moved nearly twice as fast as cells in regular medium. This speed increase was preceded by striking morphological and functional changes – cells in viscous medium increased their spread area and became flatter, displayed explosive remodeling of focal adhesions (FA), and generated stronger traction forces. We found that the response was not binary; rather, cells were able to tailor their response to the degree of viscosity. After characterizing these behaviors and examining cytoskeletal dynamics, membrane ruffling at the lamellipodia was identified as the viscosity sensor that regulates downstream changes in morphology and motility. [000182] RESULTS [000183] Elevated viscosity enhances single cell motility [000184] To simulate viscous ECF, high molecular weight hydroxypropyl-methylcellulose (MC) was added to culture medium at concentrations ranging from 0.25% to 2%. MC is an inert thickening agent that has been used as a supplement in stem cell culture medium (22) and in actin gliding assays (23) to increase fluid viscosity. These MC-supplemented media were characterized using a rheometer (FIG.6A). The highest concentration used in this study, 2% MC, increases viscosity by three orders of magnitude (FIG.6B), creating a solution with a consistency similar to that of honey. The motility of MDA-MB-231 breast cancer cells was then examined in regular versus viscous medium. Surprisingly, cells in viscous media exhibited speed increases of up to 2-fold (FIG.1A). The increases were graded, with higher viscosities corresponding to faster speeds until a plateau was reached. Cell speed during the transition from regular to viscous medium was then inspected and it was found that speed began increasing instantaneously upon the addition of viscous medium before reaching a new steady state after approximately 30 minutes (FIG.1B). Despite the increase in speed, cell movement remained random, and the directional persistence of cell migration was not significantly altered by viscous medium (FIGS. 7A, 7). Cells immersed in viscous medium continued to move faster for at least 24 hours (FIGS. 1C, 1D), indicating a prolonged effect of elevated viscosity on cellular behavior. [000185] It was next sought to verify that enhanced cell motility in MC-medium was due to elevated viscosity, rather than some chemical property of MC or the slightly increased osmolarity of the medium. The speed of cells immersed in medium supplemented with 1%poly(ethylene oxide) (PEO) was then measured, which increased the viscosity to 160 cP, similar to that of 0.75% MC. A 1.4-fold migration speed increase in cells in 1% PEO was observed, providing evidence that the cell speed increase is not polymer-specific (FIG.1C). Cell speed was also measured in medium supplemented with 1% dextran, where the osmolarity was similar to 1% MC but the viscosity was comparable to regular medium. Cells in 1% dextran did not exhibit any speed increase (FIG.1C). These results show that the cell speed increase can be attributed to elevated viscosity alone – it is not a result of changes in chemical composition or osmolarity of the medium. [000186] To determine whether the effect of viscosity on cell motility was similar across cell types, the speed of NIH 3T3 fibroblasts was measured in viscous medium. The same trend of sustained speed increases was observed in 1% MC and 1% PEO with no change in motility in 1% dextran (FIG.1D). A similar speed increase was seen after treating RAW 264.7 macrophages with 1% MC (FIG.8A) and found that elevated viscosity induced faster migration in primary fibroblasts collected from patients with wound healing disorders as well (FIG.8B). These observations provide evidence of the existence of a common mechanism across cell types allowing cells to respond to changes in viscosity. The fact that cells were able to move faster in increasingly viscous medium was counterintuitive – increased resistance should have led to a decrease rather than increase in cell speed. Indeed, a model of crawling cells assuming no adaptive changes in cytoskeletal dynamics predicted that increasing viscosity would slow down cell motility (24). This led to the hypothesis that cells were able to actively sense and respond to changes in ECF viscosity. [000187] Cells spread rapidly in response to elevated viscosity [000188] Because the cell speed increase began right after the change to viscous medium (FIG.1B), timelapse images of cells were recorded during the transition from regular to viscous medium and examined parameters known to affect cell motility, including spread area, FA dynamics, actin flow, and traction forces (25). First, interference reflection microscopy (IRM) was used to examine the spread area of MDA-MB-231 cells on the substrate, and it was found that the area began increasing immediately following medium change (FIG.1E). This increase in cell spread area continued for approximately 20 minutes before plateauing (FIG.1F). Inspection of timelapse 3D images revealed that as cells spread more, they also became flatter (FIGS.9A-9F). The same trend was observed in HEK 293 cells, further supporting the notion of a viscosity- sensing mechanism common across cell types. [000189] Examining cell spreading at various viscosities revealed that, like cell speed, the increases in spread area were graded, with higher viscosity inducing more spreading. The most viscous medium, 2% MC, caused an increase in average cell spread area of more than 2-fold (FIG.1F). The instantaneous onset of cell spreading and the viscosity-dependent maximum speed and spread area implied a sensing mechanism in response to increases in viscosity. To test whether cells were equally able to sense a reduction in viscosity, the cells were gently diluted, and 1% MC was removed before replacing it with regular medium. An immediate decrease in spread area was observed that continued for 20 minutes before leveling off (FIG.1G). Complete removal of the viscous medium was technically not possible, and the residual MC likely rendered the replenished medium slightly viscous, which could explain the small discrepancy between initial and final spread area. Plotting the viscosity- dependent speed increases against spread area increases, we found a strong linear relationship (FIG.1H), which is in agreement with a previously proposed model predicting that larger spread area and cell flattening increase speed (26). [000190] Membrane ruffling is essential for the cellular response to viscosity [000191] In the regions of the cell that spread in response to elevated viscosity it was observed that membrane ruffles were suppressed; inversely, in regions where no ruffling had been observed in regular medium, no spreading was detected after changing to viscous medium (FIG.2A). Membrane ruffling and lamellipodial protrusion are hallmarks of mesenchymal cell motility, and cycles of protrusion and retraction and ruffling are thought to facilitate haptotaxis and durotaxis (27). To measure ruffling quantitatively, MDA-MB-231 cells with eGFP-F-tractin to label F-actin and acquired z-stack images in time lapse. Peripheral membrane ruffles, which occur when protruding lamellipodia detach from the substrate, are actin-rich structures that move upward in the z-axis and then inwards. Because the membrane ruffles spanned multiple focal planes, the ruffles were segmented by projecting the eGFP-F-tractin fluorescence into a single composite image, where the signal was thresholded and integrated to represent the extent of ruffling. It was observed that membrane ruffles were suppressed to a greater degree with increasingly viscous medium (FIG.2B). Like the increase in spread area, the change in ruffling was reversible, and it recovered within approximately 10 minutes of replacing 1% MC withregular medium (FIG.2D). Plotting both the normalized cell spread area and degree of membrane ruffling over time, it was found that the two exhibited an inverse relationship (FIG. 2C). A steep decrease in ruffling preceded a more gradual increase in spread area in 1% MC. [000192] It was next explored whether there was a direct, causal relationship between the reduction of membrane ruffles and the increases in cell spreading and speed in viscous medium. To test this, fish keratocytes were used, which are well-characterized, highly motile cells with negligible membrane ruffling. Keratocytes treated with 1% MC did not exhibit any changes in spread area (FIG.2F) or cell speed (FIG.2E), agreeing with previous studies (20, 28). It was observed that a very small population (< 2%) of atypical keratocytes did exhibit ruffling and were stationary; following the addition of 1% MC, ruffling in these cells decreased and they showed large increases in spread area and motility. To further confirm that membrane ruffles were critical for viscosity-dependent cell spreading and speed increases, MDA-MB-231 cells were treated with wheat germ agglutinin (WGA), which has been shown to inhibit membrane ruffling(29). WGA treatment effectively inhibited membrane ruffling in regular medium, and subsequent addition of 1% MC did not induce any changes in spread area (FIG.2G) or speed (FIG.2H). These results demonstrated that membrane ruffling was critical for the increases in cell spread area and cell speed in response to viscosity. [000193] Elevated viscosity enhances focal adhesion formation and accelerates turnover [000194] Next was to identify the link between the changes in membrane ruffling and spread area at high viscosity by examining the role of integrin, a key molecule in cell adhesion and migration, also required in ruffle formation. Without extracellular matrix (ECM) proteins absorbed onto the substrate surface to facilitate integrin-ECM binding, cells did not display significant ruffling, and did not increase spread area in response to elevated viscosity (FIG.3A), implying that ruffling and the response to viscosity were mediated by integrin. To further confirm that integrin engagement was critical for viscosity-dependent spreading, glass-bottom dishes were coated with poly-L-lysine so that cell-substrate adhesion was mediated via electrostatic interaction s(30) rather than integrin engagement. As expected, MDA-MB-231 cells adhered to the poly-L-lysine-coated surfaces exhibited minimal ruffling and did not increase spread area upon the addition of 1% MC (FIG.10).[000195] Because membrane ruffling is a result of retrograde forces overcoming the strength of cell- substrate adhesion at the lamellipodia and pulling the leading edge rearward (27, 31, 32), it was investigated whether FAs, which facilitate cell-substrate adhesion, were affected by elevated viscosity. Timelapse images of MDA-MB-231 cells transfected with mEmerald- talin, a protein recruited early during the formation of nascent adhesions, were recorded. Inspecting the changes in mEmerald-talin intensity between subsequent frames, immediate redistribution of talin to the expanding lamellipodia following the addition of 1% MC was observed (FIGS.3B, 3C), providing evidence of extensive nascent adhesion formation. Significant changes in FA assembly and disassembly rates were also observed (FIG.3D). The assembly and disassembly rates were both approximately 25% higher in 1% MC than in the regular medium. The average adhesion size decreased by 30% in viscous medium (FIG.3F, while the total number of adhesions per cell increased slightly (FIG.3G), leading to an overall reduction in total FA area per cell (FIG.3E). Efficient FA remodeling and turnover are necessary for cell motility, and faster FA turnover has been shown to enhance cell motility (34, 35). Furthermore, FA size governs cell speed, with optimal FA size leading to faster migration (36). These results provide evidence that viscosity-dependent cell spreading is mediated by integrin and that the cell speed increase in viscous medium is a product of increased FA turnover and tuning of FA size. [000196] Actin polymerization is indispensable for viscosity-induced cell spreading; myosin II contractility is not [000197] FA-mediated cell motility is driven by actin polymerization. Actin filaments continuously grow towards the leading edge, which both drives protrusion and causes filaments to undergo retrograde flow, the rate of which decreases when actin filaments are coupled to the substrate through FAs (37). The rapid cell spreading in viscous medium implied a decrease in the rate of retrograde flow and an increase in actin-driven protrusion. It was first tested whether actin polymerization was involved by treating MDA-MB-231 cells with 100 nM latrunculin A (latA), a drug that inhibits F-actin polymerization, before immersing them in 1% MC. Viscosity-induced spreading in latA-treated cells was significantly attenuated (FIGS.4A, 4B), confirming that actin polymerization was necessary. Examining kymographs of the leading edge in cells transfected with eGFP-F-tractin, a 10-fold reduction was found in retrograde flow speed for cells in 1% MC (FIGS.4C, 4D), indicating coupling of actin to FAs to slow retrograde flow and drive protrusion.[000198] To evaluate the role of myosin contractility in viscosity-induced spreading, w cells were treated with the myosin II inhibitor blebbistatin (20 μM). After the addition of 1% MC, the blebbistatin-treated cells showed a trend very similar to untreated cells, with rapid spreading ultimately leading to a 1.7-fold increase in cell spread area (FIG.4E). These results demonstrated that cell spreading in viscous medium was independent of myosin contractility, and instead proceeded through reinforcement of cell-substrate adhesions and engagement of the molecular clutch, which slowed down retrograde flow and led to actin- driven protrusion at the leading edge. This agrees with a recent study in which lamellipodial spreading was found to be dependent on nascent adhesion stabilization and independent of myosin (38). As viscous medium suppresses membrane ruffling at the cell edge, prolonged contact between the lamellipodia and substrate could allow for integrin- ECM engagement and nascent adhesion formation, a critical early step in the formation of FAs. Indeed, it has been shown that temporal maintenance of contact between lamellipodial protrusions and the substrate facilitates nascent adhesion formation and growth (32). [000199] Cells at elevated viscosity generate stronger forces [000200] The rates of FA assembly and disassembly increased at elevated viscosity, and both processes are force-dependent (7, 39, 40). To examine whether faster FA turnover in response to increased viscosity was a result of increased force generation, continuum traction force microscopy was performed (41). It was found that total traction forces were approximately 2-fold higher when cells were immersed in 1% MC, and maximum traction stresses increased significantly as well (FIGS.5A, 5C, 11A). Traction forces transmitted to the substrate via integrin at the single-molecule level were examined using tension gauge tether (TGT) (42). Timelapses of cells on TGT-coated surfaces showed a 2.4-fold increase in TGT ruptures following the addition of 1% MC (FIGS.5D, 5E). This result demonstrated extensive engagement of integrin molecules transmitting forces greater than the 54 pN rupture threshold and suggested dynamic and extensive coupling between integrin and the actin cytoskeleton. [000201] Inhibition of myosin II contractility by blebbistatin decreased cellular forces but did not abolish the increase in traction force induced by 1% MC (FIGS.5B, 5C, 11A and 11B). This agrees with a previous finding that traction force magnitude correlates with changes in cell spread area (43), as both blebbistatin-treated and untreated cells showed increases in spread area and traction forces in viscous medium. In agreement with the findings that blebbistatin did notprevent increases in spread area or traction force generation in viscous medium, blebbistatin- treated cells still increased speed by 1.5 fold in 1% MC, an increase comparable to that of the control group (FIG.5F). [000202] Interestingly, observing cells labeled with mApple-myosinIIA, a lamella-wide distribution of myosin filaments emerged as cells spread in 1% MC (FIG.12). Some of myosin filaments formed structures known as “myosin stacks” (44), which were absent in the cells prior to the addition of the 1% MC. So, although myosin II contractility was dispensable for the response to viscosity, these data indicate that myosin could play a structural role in viscosity- induced rearrangement of the actin cytoskeleton. [000203] Modeling cell spreading in response to viscosity [000204] A mathematical model was developed to describe the dynamic changes to the cell upon increased viscosity, incorporating measurements of cell spread area, leading edge protrusion, FA area, FA assembly / disassembly rates, actin retrograde flow, and traction force. The following principles known to govern cytoskeleton mechanics (detailed further in Methods) were implemented in the model (45). First, tension resists protrusion at the leading edge, resulting in actin retrograde flow; second, forces exerted by the retrograde flow facilitate nascent adhesion formation and subsequent FA growth; third, FAs exhibit centripetal growth as a reaction-diffusion-convection process, driven by retrograde flow; and finally, strong integrin- ECM engagement promotes leading edge protrusion against cortical tension, where increasing tension causes membrane buckling and ruffling. Mapping the data to the parameter space, it was found that increased FA assembly / disassembly rates as seen in 1% MC could lead to three possible scenarios immediately following an increase in viscosity: decreased protrusion (Δl < 0) and increased FA area (ΔFA > 0); Δl > 0 and ΔFA > 0; Δl > 0 and ΔFA < 0. This model predicted that only if cortical tension decreased upon a viscosity increase would the experimental observation of Δl > 0 and ΔFA < 0 be possible (FIG.5G). [000205] This was tested by measuring cortical tension using laser ablation, a technique in which tension stored in the cortex is reported by the distance and rate of actin arc recoil following ablation (46). Confirming the prediction of the model, cortical actin arcs recoiled only half as far after ablation during the 15 minutes following 1% MC addition, representing decreased cortical tension (FIGS.13A-13E). Notably, after 20 minutes in 1% MC, coinciding with the plateau in cell spreading, cortical tension returned to the level observed in DMEM. Theobservation agreed with a previous report that cell spreading was associated with an initial drop followed by recovery in cortical tension as cells spread and available membrane reservoirs were depleted (47). Cross-examining the dynamics of ruffling and cortical tension in cells subjected to 1% MC, it was found that ruffling began to decrease instantaneously, whereas cortical tension dropped significantly only after a 10-minute delay (FIG.13C), indicating that ruffle suppression occurs prior to cortical tension reduction. [000206] Additionally, the model predicted that, to achieve a 2-fold increase in traction force in 1% MC, there must be an increase in traction force associated with nascent adhesion formation in addition to the force transmitted through mature FAs. To test this prediction of increased nascent adhesion formation, we measured changes in nascent adhesion integrated intensity upon increased viscosity and observed a rapid increase in nascent adhesion formation (FIG.3C) amounting to 40% increase in nascent adhesion formation in 1% MC compared to that in DMEM (FIG.3C), consistent with the predictions of the model. [000207] Membrane ruffling acts as a sensor of extracellular viscosity [000208] Because the presence of membrane ruffling in regular medium dictated whether or not spread area and speed would increase in viscous medium, it was reasoned that suppression of ruffling triggered these downstream responses. It was hypothesized that, at elevated viscosity, the retrograde forces of contractility and membrane tension that drive ruffling, buckling the lamellipodium and moving it upward and rearward (27, 48, 49), would be insufficient to overcome the hydraulic resistance imposed by the medium. Simulations were performed to evaluate the forces required to move the lamellipodium upward and rearward, and it was found that the drag force on a ruffling membrane increased by two orders of magnitude in 1% MC (FIGS.5H, 14A-14E). Upon the viscosity increase, the forces driving ruffling are counteracted by the drag force, which increases proportionally to viscosity. Because of the decrease in net force on the membrane, the membrane can no longer be displaced upward and rearward to form ruffles to the same extent as in regular medium. Indeed, an inverse relationship was observed between viscosity and ruffling (FIG.2B), supporting the notion that higher hydraulic resistance suppresses ruffling more effectively. As a result, the membrane remains in close contact with the substrate, leading to increased nascent adhesion growth at the leading edge; indeed, it has been shown that temporal maintenance of lamellipodial protrusions stimulates nascent adhesion formation (32). The membrane is then pushed forward more efficiently by actin polymerizationas actin filaments are coupled to new adhesions, causing a reduction in retrograde flow and an increase in cell spreading. This increase in cell spread area leads to higher traction force generation, which in turn accelerates FA turnover and tunes FA size, driving faster cell migration. [000209] The simulations showed the vertical drag force on a ruffling membrane with an area of 4x8 μm2to be approximately 1 pN in 1% MC, and the lateral drag force on a protruding membrane to be of the same order of magnitude, agreeing with previous reports (50). In advancing lamellipodia, cell-generated protrusive forces are more than sufficient to overcome this level of resistance, as the force generated by just a single polymerizing actin filament is >1 pN (51), and actin-driven cell protrusions have been reported to generate forces on the order of ones to hundreds of nN (52, 53). The force generated by polymerizing actin filaments in the 4x8 μm2model protrusion would therefore greatly exceed the sub-pN drag force. However, this force generation machinery is confined in very thin, sheet-like lamellipodia at the protruding edge of the cell, and these forces are oriented almost entirely in the xy-plane (FIG.14D). Similarly, the retrograde forces that cause out of plane buckling and lead to ruffling are primarily oriented in the xy-plane, and the component of these forces oriented along the z-axis causing ruffling is therefore extremely small. It is also known that viscous drag will significantly affect buckling at extremely low Reynolds number, which applies to motile cells and cell-scale filaments and sheets in viscous fluids (54–57), and that the force required to cause buckling increases proportionally with the coefficient of viscous drag (see Methods and FIG.14E). Therefore, while the increased drag force is easily overcome in the xy-plane by actin- driven protrusion, it is sufficient to hinder ruffling in the z-direction. [000210] The results herein, provide evidence that ruffles act as a mechanosensor of ECF viscosity instead of a passive membrane reservoir. A known example of membrane structure serving as mechanosensor is caveolae, which sense membrane tension and release membranes upon tension increase (58). [000211] To further confirm ruffles indeed actively sense and respond to the changes in viscosity, instead of passively storing and releasing membrane, membrane availability was perturbed by other means. Cells were treated with 80 μM dynasore to suppress endocytosis, inhibiting the membrane recycling from the plasma membrane to the cytoplasm, effectively rendering more membrane available that can be added to the cell surface (59, 60). No spreadingwas observed following treatment, and cells continued to exhibit ruffling. Subsequent addition of 1% MC increased the spread area as untreated cells did (FIG.15A). Similarly, no increase in spread area was observed when treating cells with hypertonic medium (530 mOsm), which increased membrane availability stored in membrane invaginations (e.g. caveolae). Subsequent addition of hypertonic 1% MC resulted in spread area increase (FIG.15B). In summary, increasing membrane availability by means other than increasing viscosity did not affect ruffling and spread area; ruffles specifically responded to viscosity change and increased spread area. [000212] Cells were then treated with hypotonic medium (160 mOsm) to increase cell volume and thereby membrane tension. A slight decrease in spread area was observed as cells swelled (FIG.15C), but cells continued to exhibit ruffling, agreeing with previous reports (61). The hypotonically treated cells were then immersed in hypotonic 1% MC, and spread area increase was observed as cells in isotonic 1% MC would, though the spreading process was relatively slower due to the elevated membrane tension from osmotic swelling (49, 62). Notably, when the hypotonically treated cells were immersed with hypotonic 2% MC, instead of 1%, larger and more rapid increase in cell spread area was observed (Supplemental FIG.15C). Because increasing the available membrane by hypertonic treatment or endocytosis inhibition, as well as releasing the available membrane by hypotonic swelling, did not result in significant changes of cell spread area like viscosity changes did, it was concluded that ruffles are not a passive reservoir of membranes, but a sensor responsive to viscosity changes. [000213] To summarize, suppression of ruffling at high viscosity initiates the following: enhanced integrin-substrate engagement, increased nascent adhesion growth, reduced actin retrograde flow, increased cell spreading, greater traction force generation, faster FA turnover, and ultimately faster cell speed. In effect, membrane ruffling acts as a sensor of ECF viscosity. [000214] Conclusion [000215] In this study, the responses of cells subjected to high viscosity was characterized, and it was found that membrane ruffling serves as a sensor of viscosity. It was concluded that the cellular response to viscosity is mediated not by a molecular sensor, but by a biophysical mechanism – an actively ruffling membrane probing the viscosity of the ECF. This hypothesis is supported by the observation that cells with negligible or inhibited ruffling did not respond to viscosity. Given the variability of ECF viscosity in vivo, membrane ruffling may have an important role in regulating cell motility and force generation, both of which are significant indisease progression (63). Whether membrane ruffling serves as a viscosity sensor for cells embedded in 3D matrices has yet to be explored, but it was noted that membrane ruffling has been observed in fibroblasts in 3D collagen matrices (64). The study focused on the effects of elevated viscosity on FA-mediated cell behavior, and the effects on other modes of cell motility, such as amoeboid movement (65) or nuclear piston-based migration (66), are still unknown. It is also possible that elevated viscosity could lead to changes in gene expression at longer timescales, as this study explored the instantaneous biophysical responses to viscosity. Further work is necessary to comprehensively understand how viscosity affects cell behavior across different timescales, modes of motility, and environments, but it is clear that the viscosity of the extracellular fluid plays an important role in regulating cell behavior. [000216] EXAMPLE 2: [000217] MATERIALS AND METHODS. The following materials and methods were used in this and other Examples. [000218] Cell culture, transfection, and drug treatment [000219] MDA-MB-231, NIH 3T3, HEK 293, and RAW 264.7 cells were cultured in DMEM (1X) [+] 4.5g / L D-glucose, L-glutamine [+] 110mg / L sodium pyruvate (Gibco), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S). AG1942 human fibroblasts with the non-wound healing phenotype were purchased from the Coriell Institute and were cultured in DMEM supplemented with 15% FBS and 1% P / S. Keratocytes were harvested from Amatitlania nigrofasciata as previously described (67), and cultured at room temperature in RPMI 1640 with 10% FBS and 1% P / S. Experiments involving animals were performed under the oversight of and in accordance with the guidelines established by the Johns Hopkins University Animal Care and Use Committee. [000220] Cells were transfected using Lipofectamine 3000 with the standard protocol provided by ThermoFisher for transfecting MDA-MB-231 cells. Cells were plated for imaging 24 hours after transfection. The plasmids eGFP-F-tractin and mEmerald-Talin were gifts from Clare Waterman, and mApple-myosinIIA was acquired from Addgene. For myosin staining, anti- myosin heavy chain antibody (BioLegend 909801) was used at a dilution of 1:500. [000221] For drug inhibition experiments, the drug concentration and duration of pretreatment are specified in the text. For all experiments involving changing medium, the same concentration of drug was added to both regular and viscous medium. Latrunculin A waspurchased from Abcam (ab144290). Photostable para-aminoblebbistatin was purchased from Motorpharma (Cat. No. DR-Am-89). [000222] Preparation of viscous medium [000223] Viscous medium was prepared by adding the indicated percentage by weight / volume of thickening agent to pre-warmed culture medium. Poly(ethylene oxide) powder, average Mv~5,000,000 (PEO); alginic acid sodium salt medium viscosity (alginate); and dextran 70,000 Da (dextran); were acquired from Sigma-Aldrich. METHOCEL J75MS, hydroxypropyl methylcellulose (MC) was acquired from DuPont. The medium was then placed onto a rotating mixer for 24 hours at 15 RPM. The viscosity measurements of the viscous media were performed at 37o C using an Anton Paar MCR 302 rheometer with a cone-plate probe and rotational tests for varying shear rates. [000224] Cell motility assays [000225] Cells were seeded at low density onto uncoated glass bottom dishes in medium containing 10% FBS. Cells were allowed to adhere for 5 hours before replacing with either viscous medium or fresh medium for control experiments. Depending on the experiment and as indicated in the text, cells were imaged either immediately after changing medium, after a short incubation of 2 hours, or after overnight incubation. Timelapses were acquired for a duration of 5 hours at 2-minute intervals with a 5X objective and a Leica DMC5400 camera mounted to a Leica SP8 confocal microscope. [000226] Single-cell tracking was performed in a semi-automated fashion using CellTracker (68), a Matlab program designed for the task, and cell motility is reported as the average speed per cell across the duration of the timelapse. Persistence and directionality analysis was performed using the DiPer (69) Excel program. [000227] Timelapse imaging and confocal microscopy [000228] All imaging was performed with environmental control at 37°C and 5% CO2. Unless otherwise noted, images were captured with a Leica TCS SP8 laser scanning confocal microscope with a 63x 1.40 N.A. Plan Apo oil immersion objective lens. Illumination was provided by a white light laser and images were captured with a Leica Hybrid Detector (HyD). Myosin stack imaging was performed on fixed cells on a Nikon N-SIM microscope equipped with a 1.49 NA 100X objective lens and an EMCCD camera.[000229] For cell spread area and FA timelapses, cells were imaged at 1-minute intervals for 10 to 30 minutes in regular medium and for 30 to 60 minutes after adding viscous medium. To test the involvement of integrin-ECM binding in viscosity sensing, cells were plated on uncoated glass dishes in serum-free medium to prevent the adsorption of ECM molecules present in serum onto the glass surface of the culture dishes. Medium changes were performed by gently removing medium from the dish with a plastic pipette and replacing with viscous medium. [000230] Image processing and analysis [000231] Cell area was determined by semi-automated threshold-based segmentation of timelapses of single cells using Fiji and Matlab. Analysis of FAs was performed using the Focal Adhesion Analysis Server (70) to determine adhesion size and area, number, and assembly and disassembly rates, and data was plotted and analyzed in GraphPad Prism 7. Nascent adhesion growth was determined by quantifying the changes in talin signal between subsequent frames. Subtracting one frame from the next left fluorescent signals only in places where talin had newly localized since the previous frame, indicating nascent adhesion growth. The integrated intensity of these signals corresponded to the relative degree of overall nascent adhesion growth. [000232] To quantify membrane ruffling, confocal z-stacks of fluorescent F-tractin were acquired at 1-minute intervals. The summed projection of these stacks created a strong signal in the tall, actin-rich ruffles, and this signal became weaker as the ruffles changed location or flattened. For each projection, a threshold was manually selected to include membrane ruffling across all frames while excluding other actin structures. The ruffle index (arbitrary unit) was then generated by integrating the thresholded area and normalizing each frame to the mean ruffle area of all the frames captured in DMEM (FIG.16). [000233] To measure nuclear height, width, and volume, cells were stained for 15 minutes with 2 μM Hoescht 33342 before rinsing and replacing with culture medium. Z-stacks were acquired just before the addition of 1% MC, and then at 10 minute intervals following addition for 30 minutes. At each time point, nuclear height and width were determined by slicing the nucleus along the long axis and taking bounding box dimensions of nuclear cross sections. Nuclear volume was determined by thresholding each slice and summing the thresholded voxels. [000234] Plots were prepared and statistical analysis was performed in GraphPad Prism 7. For plots with more than two groups, an Ordinary one-way ANOVA with either Tukey’s test formultiple comparisons or Dunnett’s test was used. Sample sizes and p values are detailed in the text and figure captions. [000235] Traction force microscopy and analysis [000236] Traction force microscopy (TFM) was performed as previously described (41, 71). Briefly, 16.3 kPa polyacrylamide substrates (12% acrylamide and 0.15% bis-acrylamide) with a thickness of 20 μm were labeled with 40 nm red and far-red fluorescent beads, covalently attached to coverslips, and coated with human plasma fibronectin (0.25 mg / mL). Cells stably expressing F-tractin-GFP were cultured on the TFM substrates overnight and placed into a magnetic imaging chamber (Chamlide) on the day of the experiment. Multi-color images of F- tractin-GFP and fluorescent beads were acquired by a spinning disk confocal microscope equipped with a 60X water immersion objective (NA 1.2, CFI Plan Apo VC, Nikon Instruments). After the image acquisition, 1% SDS was added to the imaging chamber to remove the cells, and images of the relaxed substrates were acquired. The traction stress field and integrated traction force were computed by using a custom build MatLab script as previously described (41, 71). [000237] Perturbation of membrane reservoirs [000238] Hypo / hypertonic solutions were prepared as previously described (72). Briefly, hypertonic medium was prepared by adding 200 mM sucrose to complete medium, and hypotonic medium was prepared by diluting complete medium with 50% cell culture grade water with 1 mM CaCl2and 1 mM MgCl2added. Hypo / hypertonic viscous solutions were prepared by first making hypo / hypertonic medium and then adding MC and mixing as normal (. For inhibition of endocytosis with dynasore (Sigma- Aldrich, D7693), cells were incubated in medium containing 80 μM dynasore for 2 hours before imaging began. [000239] Cortical tension measurement by laser ablation [000240] Laser ablation of actin arcs was performed as described previously (46). Actin arcs on the dorsal surface are distinct from ventral stress fibers and are less mechanically coupled to FAs and therefore tension within actin arcs should be more dependent on actin cortex tension. While stress fibers have been considered distinct structures isolated from the rest of the cortex, recent works combining theory and experiments have demonstrated that stress fibers are mechanically integrated with the cortical membrane (73, 74). This was supported by laser ablation experiments in which the cut ends of a stress fiber were observed to remain undertension after ablation, suggesting that tension within a stress fiber is also dependent on tension in the adjacent cortical regions (74). It was also observed that increases in tension through pulses of myosin-II contraction in the cortex were sufficient to drive the formation of stress fibers (73). Moreover, it was shown that the formation and maintenance of actin arcs also depend on cortical tension (75). Based on the mechanical integration of actin arcs with the actin cortex and their dependence on cortical tension, we used the recoil velocities of actin arcs as an approximation of relative changes in cortical tension in the lamellar region. [000241] Briefly, ablation was performed on a Nikon Spinning Disk confocal microscope equipped with a 60X 1.4 NA objective and an Andor iXON Ultra EMCCD camera, provided by the Nikon Center of Excellence at Vanderbilt University. Three pre-ablation were recorded at 2 second intervals, following which ablation was performed using a dedicated 100 mW UV laser (Coherent Technologies) at 50% power, using a dwell time of 500 μs for a total period of 1 s using a Stimulation Line ROI of 2 μm length. Cells were maintained at 37°C and 5% CO2using a Tokai Hit stage incubator. Following ablation, images were acquired at 2 s intervals. To measure initial cut size and retraction rate, kymographs were created using a 3-pixel thick segmented line in Fiji. Retraction rate was calculated as the distance travelled by the two severed ends per second in the first 10 seconds post ablation. [000242] Single-molecule force measurement [000243] TGT was assembled by hybridizing a cyclo-RGDfK conjugated DNA strand and biotinylated strand. The sequence (18 base pairs) was taken from previous work(76). For real- time force detection, quenched TGT (qTGT) with a Cy3-BHQ2 quencher pair was used(77). Coverslips were biotinylated and passivated by PEGylation to minimize nonspecific bindings. The surfaces were coated with neutravidin and qTGT (1 μM) sequentially. Biotinylated gold nanoparticle was immobilized to the surfaces for the drift correction. Cells were seeded on the surface and incubated for an hour in DMEM before measurement. A Nikon Eclipse Ti microscope equipped with a custom total internal reflection fluorescence (TIRF) microscopy module (543 nm laser and custom filter set) was used for live-cell imaging. Time-lapse images were obtained at one-minute time interval before and after the medium change (1% MC). [000244] To reconstruct the real-time force map, we first performed subpixel drift correction. Then, qTGT rupture events at each time point were calculated by subtracting subsequent frames in the timelapse. The sum of rupture events was projected for 20 minutes inDMEM and for 20 minutes in 1% MC to generate cumulative rupture maps for each condition. The integrated intensity of these rupture maps reported the total amount of rupture event in each condition. To account for noise, a background ROI of the same size as the cell ROI was manually identified and the integrated intensity of background ROI was subtracted from the cell ROI. [000245] Simulation of membrane ruffling and estimation of drag force [000246] To mimic the motion of membrane ruffling and protrusion, we adopted a finite volume method (FVM) using ANSYS-CFX 14.0, a commercial CFD software. A cuboid of 4 μm x 8 μm x 0.5 μm was constructed to model the membrane and was set as a rigid immersed solid body – the moving boundary of an immersed solid. For the fluid domain, a 50 μm x 50 μm x 50 μm cube was constructed and the fluid wall was set as a free slip condition. The number of nodes and tetrahedral elements were 4,198 and 21,918 for the membrane domain and 4,816 and 26,196 for the fluid domain, respectively. For ruffling, the cuboid domain was designed to be rotated on the rotation axis with 0.05 rad / s of angular velocity. For protrusion, the cuboid domain was designed to move in one direction with 0.2 μm / s of translational velocity. [000247] To describe fluid motion, two equations were utilized – mass (continuity equation) and momentum conservation (Navier-Stokes equation) with assumptions of an unsteady and 15 incompressible flow and no body force. The governing equations are as follows (78): [000248] where u, ρ, p, and μ arevelocity components (u, v, w), fluid density (i.e., DMEM or 1% MC), pressure, and dynamic viscosity, respectively. The fluid density is 1 cP for DMEM and 450 cP and 1% MC. The time step and total time for unsteady analysis were set to be 1 s and 20 s, respectively. The high-resolution discretization and second-order backward Euler discretization were utilized as an advection scheme and transient scheme, respectively. Total calculation time per model ranged from 30 min to 1 hr. The root mean square (RMS) residuals indicating local discrepancy of conservation equation per control volume were used by setting convergence criteria as 10-4.[000249] After simulating membrane and fluid motion, to calculate the total drag force in the vertical direction, the drag force equation, composed of pressure drag force and skin friction drag force, was leveraged as follows (79): shear stress (WSS), n the normal direction to thearea to the surface with area dA, and i the unit vector inthe vertical direction. [000251] Governing equations for a beam under compressive loads in a viscous fluid [000252] Consider an Euler-Bernoulli beam under a spatially constant compressive force ^^ ^^( ^^ ^^). The deformation of the beam is described by its vertical deflection ^^ ^^( ^^ ^^, ^^ ^^) at any point ^^ ^^ along its length. Equilibrium of forces in the vertical direction, accounting for viscous forces but ignoring any inertia terms, is given by:[000254] The data has shown that extensive cell spreading accompanies the enhanced cell migration in the viscosity response. Although myosin II contractility is not essential, actin polymerization and FA are indispensable for this cellular response. This precipitates the mechanistic question of how FA centripetal growth and actin polymerization contribute to the cell spreading. Addressing this question will allow us to understand how membrane ruffles modulate the FA-actin axis in response to the elevated viscosity. It was hypothesized that FA anchors the actin polymerization to protrude the cell front, which is held back by the cortical tension. It is the balance between FA-mediated actin protrusive force and the cortical tension that sets the extent of cell spreading.[000255] To test this hypothesis, we leveraged mathematical modeling to explore how the mechanistic interplay between FA centripetal growth, actin polymerization, and cortical tension drives the cell spreading. Built upon our previous modeling effort (45), this new model is aimed to specifically capture the essence of the FA-mediated viscous response of cell spreading. The FA centripetal growth was modelled as a reaction-diffusion-convection process, driven by the retrograde actin flux. The resulting force balance between the cortical tension and the FA-actin flux mechanical engagement sets the extent of cell front protrusion. Since the experiments herein demonstrated that myosin II contractility is not essential for the cell response to the elevated viscosity, the model does not include this factor and the associated FA maturation process. Bearing this in mind, the qualitative description of the key model elements are as follows (FIG. 17): [000256] 1. As cortical tension resists the cell front protrusion, much of the actin polymerization at the membrane ends up as the retrograde actin flux, flowing toward the cell interior. [000257] 2. When the retrograde actin flux tugs the nascent FA, this initial pulling stimulates the catch-slip bond behaviors of FA. On one hand, the pulling opens the binding sites of the adaptors, leading to recruitments of additional integrins and adaptors and, hence, engagement with additional F-actins. On the other hand, this pulling breaks the actin-adaptor- integrin-ECM linkage when it becomes sufficient large. [000258] 3. The resulting mobile FA components, while diffusing slowly, drift with the retrograde actin flux. During the drifting, some of these mobile species will rebind to ECM and deposit downstream. As these episodes of binding, pulling, breaking, drifting, and rebinding continue, the FA grows centripetally. [000259] 4. The FA engagement with retrograde actin flux anchors the actin network to protrude the cell front, against cortical tension. [000260] Taken together, the model describes the spatial-temporal evolution of FA growth in coupling to the dynamics of branching actin polymerization and cell front protrusion. [000261] EXAMPLE 3: VISCOSITY-DEPENDENT WOUND HEALING IMPAIRMENT IN DISEASE MODEL SYSTEMS [000262] Overview: We seek to characterize the changes in neutrophils, macrophages, fibroblasts, and epithelial cells when subjected to high viscosity, at both short (seconds to minutes)and long (hours to days) timescales. This is to expand on the current understanding that upon viscosity increase, cells exhibiting ruffling lamellipodia flatten themselves, increasing migration speed, with transcription factor YAP translocating into the nucleus. At the cellular level, we will examine parameters relevant to cell motility, proliferation, energetics and chromatin organization in every airway cell type. At the molecular level, we will examine gene expression, protein phosphorylation, and Ca2+concentration in every cell type. [000263] Cellular response to elevated viscosity [000264] Microtubules: Though it was shown that the polymerization of actin cytoskeleton increases in cells with ruffles (ruffling cells) at high viscosity, how microtubule cytoskeleton behaves at high viscosity is unknown. Microtubule dynamics governs intracellular trafficking of secretory vesicles containing cytokines during exocytosis, chromosome segregation during cell division, and directional persistence during cell migration. Therefore, understanding whether and how it is affected by viscosity will inform us whether and how cytokine secretion, mitosis, and directional persistence is affected, which is important for migrating cells to close the wound. This insight can afford us to utilize known microtubule-targeting drugs (e.g. taxol, nocodazole) to rescue the affected directional persistence and cell division at high viscosity. We will perform timelapse imaging of fluorescently labeled microtubule plus tips in neutrophils, macrophages, fibroblasts and epithelial cells at mucin- supplemented medium of various viscosities, ranging from 10^-3 Pa·s (water-like) to 2 Pa·s (honey- like), and measure the growth speed, shrinkage speed, catastrophe rate and rescue rate. To study the adaptation to high viscosity over time, images will be acquired 0, 30, 120, 240, 480, 720, 1440, 2160 minutes after changing the medium from regular to viscous. These four parameters collectively contribute to how fast microtubules change cellular shape, alter migration directionality and transport cytokine for secretion. Drugs inhibiting specific regulatory proteins responsible for these parameters are candidates to restore the abnormal wound healing response caused by microtubule-dependent cell adaptation at high viscosity. [000265] Chromatin: Nuclei of ruffling cells flatten as the rest of the cell body at high viscosity. Deformation of the nucleus usually leads to immediate chromatin reorganization, changing the ratio of heterochromatin to euchromatin. Heterochromatin is highly condensed chromatin, as opposed to euchromatin which loosely winds around histone proteins. Heterochromatin is less accessible to transcription factors and DNA polymerases, thus hindering DNA replication and gene transcription. If the heterochromatin to euchromatin ratio changes, the proliferation rate and gene expression profilesof cells subjected to high viscosity may change accordingly. The highly condensed heterochromatin is not accessible to DNA binding dyes such as DAPI and Hoechst, either. Therefore, we will analyze the images of nucleus at viscosities ranging from 10^-3 Pa·s to 2 Pa·s and stained with Hoechst, in neutrophils, macrophages, fibroblasts and epithelial cells. We will determine the heterochromatin content by estimating the stain-free area of the nucleus. The result can show the trend of chromatin condensation at various viscosities in each cell type. Moreover, it was recently shown nuclear deformation during cell migration specifically changes the extent of trimethylation at lysine 27 on histone H3 (H3K27me3) and acetylation at lysine 9 on H3 (H3K9ac). [000266] Cells subjected to various mucus viscosities are stained using antibody targeting H3K27me3 and H3K9ac. In addition, we will perform immunostaining for H3K4me1 / 2 / 3, all of which are also implicated in fibrosis and inflammation. The result can show inform whether H3 modification differs at various viscosities. Therapeutic agents inhibiting methylation / acylation or demethylation / deacylation may be used to restore altered proliferation rate and gene expression at high viscosity. Measurements can be performed at varying time points to assess adaptation to high viscosity over time. [000267] Proliferation: Epithelial cell proliferation can be important for reepithelization during wound healing; whereas excessive proliferations of fibroblasts and immune cells are linked to harmful fibrosis and inflammation. We will perform immunofluorescence of Ki67, a proliferation marker95,96, in neutrophils, macrophages, fibroblasts and epithelial cells at viscosities ranging from 10^-3 Pa·s to 2 Pa·s, at various time points. In addition, we will use flow cytometry to survey the cell cycle distribution in cells subjected at various viscosities at various time points. This result can establish the cell cycle context for altered proliferation rate at high viscosity. For example, larger subpopulation in S phase implies slower DNA replication potentially due to higher heterochromatin content; and larger G1 or G2 / M subpopulations imply cell cycle arrest due to perturbation in microtubule dynamics. This information can help design treatment to restore wound repair functions. [000268] Intracellular Ca2+: As ruffling cells increase the spread area upon viscosity increase, plasma membrane is rearranged by protruding actin filaments, which can change the configuration of stretch-activated ion channel, such as Piezo1 / 2 and TRPV4 (98–101), among others. Additionally, traction force increase, which has been demonstrated in ruffling cells at high viscosity, is correlated to Ca2+influx. Moreover, as a ruffling cell flattens during the process of viscosity-dependent spreading, the cell volume decreased by 10%, suggesting efflux of water and ions within the cell through variouschannels. Water efflux also changes the intracellular Ca2+concentration which regulates many biochemical reactions. Therefore, we will use live-cell imaging to determine the effect of viscosity on intracellular Ca2+concentration. We will perform ratiometric imaging of Ca2+biosensor GCaMP6 and cytoplasmic mCherry expressed in neutrophils, macrophages, fibroblasts and epithelial cells at viscosity range and various time points. If significant Ca2+concentration changes are detected, we will employ pharmacological and genetic perturbations to identify specific Ca2+channels and aquaporins activated at high viscosity. The molecules identified here will be further tested as targets to treat impaired wound healing. [000269] Molecular response to elevated viscosity [000270] Phosphoproteomics: It is likely phosphorylation events occur in pathways intersecting with the visco-sensing pathway. For example, traction forces generated by retrograde F-actin flow increases at high viscosity and may result in phosphorylation of PLCγ1, which in turn will phosphorylate many downstream substrates and potentially further amplify inflammation or fibrotic cascades in immune cells and fibroblasts, respectively. We will profile the phosphoproteomics, using mass spectrometry, in neutrophils, macrophages, fibroblasts and epithelial cells at mucin- supplemented medium of various viscosities and at various time points. We will screen for protein species with changed phosphorylation level relevant to inflammation and fibrosis. The result can help us further identify points of intervention to restore wound repair functions. [000271] Transcriptomics: Transcription factor YAP translocates to the nucleus in ruffling cell at high viscosity, and the nucleus itself flattens, possibly inducing chromatin reorganization and subsequently changing the landscape of gene transcription. It is known YAP promotes gene expression associated with neutrophil degranulation and fibroblast activation, but many more genes supporting inflammation or fibrosis can also be upregulated at high viscosity. We will profile the transcriptomics, using bulk RNAseq, in neutrophils, macrophages, fibroblasts and epithelial cells at various viscosities and at various time points. We will screen for genes with altered expression levels relevant to inflammation and fibrosis. The information gained can help us identify gene targets to be silenced to restore wound repair functions. [000272] Developing a 3D bioprinted COPD model [000273] Overview: We will construct an in vitro COPD model by 3D bioprinting, consisting of human bronchial epithelial cells, endothelial cells, fibroblasts and immune cells. Reconstituted synthetic mucus with comparable viscosity to that of COPD patients will be used to cover the airwayepithelium. Lamellipodial ruffles are observed in immune cells and fibroblasts in 3D ECM, drag force should exert the same effect on these ruffling cells and elicit similar ruffle suppression leading to speed increase, shape flattening as observed in 2D. [000274] Bioprinted in vitro airway model system [000275] Design and fabrication: The layout of bioprinted airway tissue (BAT) will be first drawn using CAD software and imported to a bioprinter for fabrication. It will consist of two layers, with epithelium at the top consisting of lung epithelial cells, and stroma at the bottom consisting of fibroblasts and endothelium-lined blood vessels to simulate microvasculature. To bioprint, cells will be first mixed with “bioink”, and then deposited at prescribed spatial locations in the culture chamber by a motorized printhead to form layers and hollow cylinders as blood vessels. Bioink is a specialized medium containing the ECM proteins enriched in the specific tissues of interest, and an inert thickening agent to increase the “printability”. For example, bioink containing collagen IV, laminins and proteoglycans, which are primary ECM proteins in the lung basement membrane, will be used to print the lung epithelium in BAT. To introduce immune cells into the system, neutrophils and macrophages suspended in culture medium will be flown through BAT, using a syringe pump at the flow rate comparable to that of microvasculature in the lung. Fibroblasts and immune cells are expected to migrate to the epithelium following chemoattractant gradients upon injury, as a response of wound healing. The BAT samples will be cultured for 3 weeks, and dynamic behaviors such as cell division and cell migration will be imaged periodically. During the weeks-long culture, we also expect capillaries sprouting from the bioprinted blood vessels. [000276] Validation of the physiological representation: To verify that BAT is biologically representative, we will first examine whether tissue organization and cell morphology are similar to native tissues. BAT will be subjected to formalin-fixed paraffin-embedding (FFPE), sectioning, and immunostaining or hematoxylin and eosin (H&E) staining. The cell density, cell size, cell shape aspect ratio, ECM composition, will be quantified using ImageJ. The diameter and the number of capillary sprouts will also be quantified. By determining the corresponding quantities in images of human airway tissue sections and subsequent statistical analysis (ANOVA), we can assess whether BAT exhibit morphological characteristics resembling in vivo conditions. Next, we will examine the gene expression fidelity of BAT via gene ontology (GO) analysis. BAT will be harvested and processed for proteomic assay using mass spectrometry. We will compare published data and the data acquired from BAT. We expect gene expression in BAT statistically comparable to that in nativetissues. [000277] Modeling COPD in BAT [000278] Viscous mucus: To mimic the typical COPD condition in which the airway is covered with viscous mucus, we will follow established protocols and use mucin protein MUC5, the primary mucin of the airway, to reconstitute mucus that is 1000-fold more viscous than normal mucus and cover the surface of BAT with the reconstituted mucus. Different viscosities can be achieved by adjusting mucin concentrations. We will reconstitute normal and highly viscous mucus to cover BAT surface. We note that even the differences in dynamic cellular behaviors are observed between viscous and normal mucus, it is possible that it is caused by the biochemical property of mucin, instead of viscosity. To rule out this possibility, viscous fluids containing other thickening polymers, such as methylcellulose, alginate, or poly-ethylene oxide, will also be tested. [000279] Cigarette smoke exposure: Cigarette smoke extract (CSE) is widely used in COPD studies because COPD develops most commonly in smokers. To induce COPD symptoms, BAT will be subjected to CSE of an appropriate dosage following established protocols, based on which we expect that epithelial shedding will occur after 1-3 days, creating wounds in the epithelium, to which neutrophils, macrophages and fibroblasts will migrate as a part of wound healing process. CSE will be applied to BAT at an appropriate frequency to induce constant shedding. Alternatively, laser ablation will be applied to BAT to create epithelial injury. [000280] Establishing an ex vivo mouse lung model for COPD [000281] Overview: We will also employ precision cut lung slices (PCLS) from mice to study the wound healing processes at various viscosities. It was shown that PCLS from smoke-exposed mice exhibit responses to virus infection and chemokine stimulation similar to COPD patients. Because PCLS contain all cell types found in the airway tissue and feature ECM remodeling associated with COPD, agreement between results from PCLS and from BAT will further validate our hypothesis that abnormal wound healing at high viscosity causes exacerbation of muco-obstructive diseases. [000282] Modeling COPD in PCLS [000283] PCLS samples: Following a previously established protocol, we will prepare PCLS from mice overexpressing IL13, which is a hallmark of muco-obstructive lung diseases. The mice will also be treated with naphthalene to create airway denudation in need for airway repair. First, the dissected lung of sacrificed mice will be inflated with agarose solution via the trachea, followed byimmersion in ice-cold buffer for rapid agarose gelation. The agarose-filled lung will then be sectioned to PCLS with the thickness of approximately 150 μm. PCLS from healthy mice will also be prepared and used as a control. [000284] Viscous mucus: We expect that viscous mucus will be spontaneously secreted by airway cells in IL13 overexpressing mice treated with naphthalene. We will measure the viscosity of the mucus collected from the mice. If the viscosity of the collected mucus is significantly lower than that associated with severe COPD, we will cover the PCLS using reconstituted mucus or other viscous fluids. [000285] Characterization of abnormal wound healing induced by high viscosity in 3D bioprinted COPD model [000286] Overview: We will quantify wound closure efficiency, inflammation and fibrosis in BAT and PCLS covered by normal and viscous mucus. By comparing the results from samples at different viscosities, and by documenting the timeline of various events relevant to COPD progression, correlation between viscosity and inflammation or fibrosis can be inferred. [000287] Longitudinal live imaging: We will image the BAT and PCLS samples longitudinally to monitor the migration and proliferation of epithelial cells, fibroblasts, neutrophils and macrophages at the wound site. To distinguish different cell types, each cell type will be labeled with spectrally distinct fluorophores. For example, before incorporation into BAT, epithelial cells will be infected with lentivirus carrying DNA sequences encoding CFP (blue), fibroblasts with GFP (green), neutrophils with mCherry (red), and macrophages with iFP (far red). Similarly, native cells in PCLS can be stained by fluorescently labelled antibody targeting cell-lineage markers: CD11 for macrophages and neutrophils, EpCAM for epithelial cells, and FAP for fibroblasts. BAT and PCLS will be imaged for 4 hours every day from Day 0 to Day 21 and continuously for 72 hours using a confocal microscope, respectively.3D timelapse images will be acquired at intervals of 5 minutes. The timelapse images will be analyzed so that the number of cells can be counted, and cell motility evaluated using cell tracking software. Proliferation rate and cell motility, will be plotted against time. We anticipate identifying correlations between viscosity and the increased number and migration speed of pro-inflammatory, pro-fibrotic cells. [000288] Gene expression profiling in fixed samples: To evaluate the changes in gene expression over the course of viscous mucus exposure, BAT and PCLS samples will be fixed at various time points. From Day 0 to Day 21, BAT samples will be fixed every three days, 8 time pointsin total. From Hour 0 to Hour 72, PCLS will be fixed every 8 hours, 10 time points in total. The fixed samples will be processed to produce FFPE sections, following by immunostaining to examine the expression of proliferation marker (Ki67), fibroblast activation (αSMA), oxida- tive stress / injury response (nitrotyrosine), cell death (activated caspase 3), and TGFβ signaling (psmad2). Fluorescently labeled antibodies against these genes and the cell lineage markers of different airway resident cell type (immune cells: CD11, fibroblasts: FAP, epithelial cells: EpCAM) will be used. The stained samples will be imaged with a fluorescence microscope and quantified for intensity for gene expression in each cell type. In addition, another batch of BAT and PCLS samples will be snap-frozen in liquid nitrogen at the time points indicated above, followed by cryostat cutting to produce 10 μm- thick sections on slides. The sections will be stained with antibodies recognizing cell lineage markers, and subjected to laser capture microdissection, where specific cell types are dissected from the section by high-power laser. The dissected immune cells, fibroblasts and epithelial cells will be lysed separately for RNAseq-based transcriptomics assay. The gene expression results acquired here will also be compared to those of single cell types. The comparison will inform us whether there is synergy between fibroblasts and immune cells at high viscosity leading to more serious inflammation and fibrosis. [000289] Statistical Analysis and Expected Outcome [000290] Statistical Analysis: At least 10 tests per experimental condition will be performed. To examine the effect of various viscosities, results from different groups will be analyzed using ANOVA followed by Tukey post-hoc tests. The values measured in different groups will be deemed significantly different with p<0.01. [000291] Expected Outcome: We expect to observe graded increase in gene expression and protein phosphorylation related to inflammation and fibrosis in neutrophils / macrophages and fibroblasts respectively as viscosity increases. We expect to observe slow re-epithelization, excessive recruitment of activated immune cells and fibroblasts, scar tissue formation in BAT and PCLS samples during wound healing at high viscosity. [000292] Preliminary Data: Using a co-culture consisting of lung epithelial cells and fibroblasts to model wound healing at high mucus viscosity, we observed that at viscosity comparable to that of normal airway, fibroblasts and epithelial cells moved at similar speed, effectively closing the wound. At viscosity comparable to that of COPD mucus, fibroblasts moved 200% faster, while epithelial cells were significantly hindered by high viscosity, moving only half as fast. As a result, the originalwounded area was populated with fibroblasts only with significantly higher ECM deposition. This result indicate viscous mucus indeed alters the cellular population at the wound site, in favor of ruffling cells capable of promoting fibrosis. [000293] Alternative Strategies [000294] During wound healing, neutrophils may undergo cell death and release neutrophil extracellular traps (NETs) consisting of nuclear DNA. While NETs further increase mucus viscosity, resulting in even acute activation in fibroblasts, it is possible that fibroblasts are activated through toll- like receptor (TLR)-mediated DNA sensing pathway. By comparing the transcriptomic data of single cell type culture and BAT, we can evaluate the likelihood of such possibility. If we find significant discrepancy between the datasets, we will repeat the experiments with a revised protocol, in which BAT and PCLS samples will be treated with DNAase to eliminate NETS, thus decoupling the effects of viscosity and TLR signaling on wound healing. [000295] Identify potential treatments for viscosity-dependent wound healing [000296] Inhibitors to known pathways involved in visco-sensing are tested, evaluating their effects in suppressing excessive inflammation and fibrogenesis. Next, we will test whether enhance the collective migration speed of epithelial cells at high viscosity can restore re-epithelization. We will also test whether mucolytic can be used in combination with the aforementioned molecules to further restore wound repair. Last, based on the result in Aim 1, we will treat neutrophils, macrophages, fibroblasts and epithelial cells with appropriate drugs and evaluate whether ion fluxes, microtubule dynamics, chromatin re-organization at elevated viscosity can be reversed and wound repairing restored. [000297] Inhibition of visco-signaling to suppress inflammation and fibrotic cascades [000298] Overview: Ruffles are the visco-sensor triggering the rapid responses upon viscosity increase, resulting in accelerated migration in ruffling cells but not epithelial cells. We will test whether inhibiting ruffle formation, thus visco-sensing, can reverse the subsequent speed increase in ruffling cells, which are recruited excessively to the wound site. We will do so by interfering with cofilin and Dia1, known to be involved in ruffle formation. [000299] Cofilin: Ruffle formation in immune cells (e.g. neutrophils) and mesenchymal cells (e.g. fibroblasts) requires an optimal concentration of activated cofilin. Too high or too low concentration abolishes the ruffles. By decreasing total coflilin concentration or increasing activated cofilin, ruffles might be suppressed. We will decrease total coflilin concentration by silencing cofilinexpression in neutrophils, macrophages and fibroblasts using RNAi. We will increase the activated cofilin sub-population by treating cells with LIM kinase (LIMK) inhibitor, a LIMK inactivates cofilin by phosphorylation, and LIMK inhibition activates cofilin. Neutrophils, macrophages and fibroblasts will be treated with cofilin RNAi or LIMK inhibitor prior to exposure to various viscosities. The key viscosity response parameters ruffling, spread area, and migration speed, will be quantified in each cell type and compared to the untreated controls. [000300] Dia1: This formin family member, Dia1, has a role in the assembly of protrusive structures, including lamellipodia and ruffles. Indeed, Dia1 depletion was shown to reduce the number of polymerization-competent actin filaments at the cell edge and interfere with the Arp2 / 3 activity leading to a dramatic decrease in protrusion and ruffle formation. Therefore, Dia1 inhibition is likely to suppress ruffle-dependent inflammation and fibrosis at high viscosity. To test this, we will quantify the suppressive effect of Dia1 depletion on ruffles in neutrophils, macrophages, and fibroblasts at relatively low viscosity (normal mucus). We anticipate Dia1 depletion decreases ruffles. We will also analyze its effect on spread area, and migration speed at various viscosities. Protruding lamellipodia are implicated in receptor signaling. In agreement, protrusion suppression in fibroblasts by Dia1 inhibition attenuates cellular response to fibrosis-promoter TGFβ, further supporting the link between visco-signaling and fibrosis. To verify, we will suppress ruffles either by Dia1 depletion in cells stimulated by pro-inflammatory cytokines (IL- 1,4,6, TNFα, TGFβ). We expect a dampened response to these cytokines where cells exhibit slower chemotaxis, less proliferation, and reduced ECM deposition in respective assays. [000301] Inhibition of YAP nuclear translocation [000302] Overview: Nuclear translocation of YAP is observed as viscosity increases. Because YAP promotes gene expression resulting in proliferation, inflammation and tissue infiltration in both macrophages and neutrophils, as well as activated phenotypes in fibroblasts, we will test whether YAP inhibition in ruffling cells can restore wound repair functions and prevent pro-inflammatory responses at high viscosity. [000303] YAP inhibition: YAP / TAZ inhibitor-1, histone deacetylases (HDACs) inhibitors (e.g. Trichostatin A), YAP-binding molecules (e.g. Verteporfin), quinolinols, and dopamine receptor D1 (DRD1) agonists (e.g. dihydrexidine, DHX) were reported to effectively inhibit YAP nuclear translocation and / or YAP-mediated transcription. In particular, it was shown that DHX reduced lung and liver fibrosis in mouse models. Therefore, we will treat neutrophils, macrophages and fibroblastswith the aforementioned inhibitors prior to exposure of various viscosities at various time points. We will examine the ratio between YAP in the nucleus and in the cytoplasm. We will also perform transcriptomic analysis. The results will be compared to the untreated controls, in order to evaluate whether YAP nuclear translocation is suppressed, and the YAP-mediated proinflammatory, fibrotic gene expression at high viscosity is attenuated. We will identify the compound of the highest efficacy, and examine its therapeutic potential in treating muco-obstructive diseases. [000304] Enhancement of collective migration for re-epithelization [000305] Overview: Epithelial cells exhibit minimal ruffles during collective migration. Without ruffle-mediated adaptation to high viscosity like fibroblasts, their migration speed decreases, leaving the wound site occupied predominantly by immune cells and fibroblasts. We will test whether introducing positive regulator of collective migration to lung epithelial cells can enhance the stalled re- epithelization at high viscosity. [000306] Integrin: Positioned between the plasma membrane and the ECM substrate, integrin governs the cell adhesion and migration through binding to and detach from the ECM molecules. Different integrin isoforms exhibit different binding affinity and dissociation rate to various ECM proteins (e.g. collagen, fibronectin). It was shown that integrin β1 and β4 promote collective migration in epithelial cells. Moreover, when pairing with β1, integrin α4 and α9 also promote collective migration. Therefore, by transfecting cells with cDNAs, we will exogenously express integrin α4, α9, β1, β4 individually or combinatorically in epithelial cells, followed by exposure to various viscosities. We will then quantify the three viscosity response parameters ruffling, spread area, and migration speed, in epithelial cells. We will also perform scratch-wound healing assay. The results will be compared to those from untreated controls. [000307] Preliminary Data: We observed that upon TGF-β stimulus, fibroblasts depleted of Dia1 do not exhibit upregulated α-smooth muscle actin (αSMA) and collagen secretion, both of which promote fibrosis. This demonstrates Dia1 depletion suppresses pro-fibrotic phenotypes. Furthermore, this suppressing effect is not caused by downregulation of TGF-β receptor (TGF-βR) on the cell surface, nor attenuation of TGF-β signaling that activates SMAD family transcription factors. Rather, such suppression is correlated with damages in mechanically overstressed actin cytoskeleton in Dia1- depleted cells. Reducing contractile force generation prevents the damage and restores pro-fibrotic phenotypes. Given that traction stress increases significantly at high viscosity, Dia1 depletion can possibly further stress cytoskeleton, resulting in substantial damages and disrupting TGF-β-mediatedfibrotic cascades in the airway of muco-obstructive lung disease patients during wound healing. [000308] Microtubule perturbation: Actin retrograde flow generates higher forces in ruffling cells at higher viscosity. The higher retrograde forces likely increase microtubule instability with higher catastrophe frequency1, resulting in faster persistent migration, directing immune cells and fibroblasts to occupy the wound site ahead of epithelial cells. If increased microtubule instability is indeed observed, we will test whether stabilizing microtubules by taxanes (e.g. taxol) can slow down ruffling cells, at high viscosity, moving towards the wound, of which the drugs were shown capable at water-like, “regular” viscosity in previous studies. [000309] Phosphorylation regulation: If protein species relevant to inflammation and fibrosis are identified with changed phosphorylation level, we will test the capacity of available inhibitors to reduce inflammation and fibrosis at high viscosity, using wound healing assays. For example, previous studies showed that increased focal adhesion kinase (FAK) and Src phosphorylation is associated with rapid focal adhesion turnover. FAK and Src phosphorylation likely increases at high viscosity where focal adhesion turnover was reported to increase, and the kinases will in turn activate their downstream substrate by phosphorylation, many of which are related to chronic inflammation and fibrosis. FAK and / or Src inhibitors might intercept these cascades. [000310] Transcription regulation: If genes related to inflammation and fibrosis are identified in 1.1.3.2 with changed expression level, we will test the efficacy of specific RNAi targeting these genes in ruffling cells in reducing inflammation and fibrosis at high viscosity by performing wound healing assays. [000311] Ca2+modulation: If intracellular Ca2+concentration increases at high viscosity is observed, and specific channels mediating such increase are identified, we will test whether ion channel inhibition or depletion can reduce inflammation and fibrosis at high viscosity by performing wound healing assays. [000312] Proliferation modulation: If faster proliferation in fibroblasts and macrophages and / or slower in epithelial cells at high viscosity are observed, we will test proliferation inhibitors (e.g. Mitomycin C) in fibroblasts and macrophages and / or promoting factors (e.g. IL1) in epithelial cells. We will evaluate whether these drugs can restore wound healing in the co-cultures of fibroblasts, immune cells and epithelial cells. [000313] Epigenetic modification: Flattening of the nucleus might change the heterochromatin to euchromatin ratio, and the abundance of H3K27me, 3H3K9ac and H3K4me1 / 2 / 3, all of which altergene expression related to inflammation and fibrosis. If chromatin reorganization can be linked to expression of proinflammatory fibrotic genes, we will test whether reversing them can suppress inflammation and fibrosis. If it is observed that acylation increases, then histone acetyltransferase inhibitors will be tested; if acylation decreases, then Histone deacetylase inhibitors; if methylation increases, then protein / histone methyltransferase inhibitors; if methylation decreases, then histone demethylase inhibitors. We note that epigenetic drugs affect the whole epigenome, casting broad impacts on gene expression, and may cause unintended effects. Therefore, even if they prove to suppress viscosity-dependent inflammation and fibrosis, epigenetics drugs will be only considered as therapy if specific treatments are unavailable. [000314] Develop specific treatment for viscosity-dependent wound healing impairment [000315] We have developed liposome-based drugs that preferably include one or more or preferably all three of the following functions: (i) mucus penetration, (ii) cell type-specific targeting, and (ii) drugs to treat epithelial cells, fibroblasts, or inflammatory immune cells. It can be significant that the drug carriers can specifically target different cell types, as drugs inhibiting cell motility should only be delivered to fibroblasts but not epithelial cells, otherwise the treatment will further impair wound healing. [000316] Fabrication of mucus-penetrating drug-carrying nanoparticles [000317] Overview: In one aspect, wound healing-improving drugs are provided by encapsulating one or more therapeutic agents in mucus-penetrating liposomes (MPLs). MPL transport and targeting specificity can bequantified to ascertain efficient drug delivery. [000318] Drug-encapsulated, cell type-specific MPLs: MPLs are surface-modified liposome capable of escaping the adhesive and steric interactions with mucin molecules, and penetrating the mucus rapidly. MPLs deliver the payload to the tissue underneath the mucus. This mucus penetrating capacity is achieved by grafting extremely dense layers of low-molecular-weight (<5 kDa) polyethylene glycol (PEG) onto the liposome surface. MPLs can penetrate viscous mucus nearly as rapidly as they diffuse in water. Synthesizing MPLs using PEG conjugated with antibody targeting cell-type markers of fibroblasts (FAP), neutrophils / macrophages (CD11), and epithelial cells (E- cadherin), cell-type specificity can be achieved. E-cadherin, localized at the lateral side of epithelial cells, is exposed to antibody conjugated to MPLs only at the wound site. By mixing drugs with lipids during synthesis, drugs can be encapsulated in MPLs. The present systems include MPLs containing one or more therapeutic agents with appropriate cell-targeting antibody. For example, for TrichostatinA (e.g. for inhibiting fibroblast activation), Trichostatin A-encapsulated MPLs with anti-FAP antibody on the liposome surface is prepared and utilized. [000319] Mobility in mucus: Fluorescently-labeled MPLs are added on top of synthetic mucus of various viscosities in a glass-bottom dish. The mucus layer can be e.g.70 μm in height, mimicking the scenario of severe mucus accumulation in human airway due to inefficient clearance in patients. 3D timelapse images will be acquired over the course of 5 hours, 30-fold longer than the normal airway clearance time (10 minutes). The images can be analyzed using the following parameters: (i) uniform dispersion: the size of fluorescent objects will be measured; (ii) mucus penetration rate: the rate at which MPLs move from the top to the bottom of the mucus layer by established single particle tracking algorithm. The measurement results can be compared to the those proven to be clinically effective in drug delivery through mucus. The measurement results can inform at which viscosity MPLs efficiently penetrate the mucus. [000320] Efficiency and specificity: A series of tests can be performeds to assess the delivery efficiency of MPLs containing different drugs targeting different cell types. First, MPLs targeting different cell types are added to single cell-type cultures of neutrophils, macrophages, fibroblasts or epithelial cells, covered with synthetic mucus of various viscosities.3D timelapse images are acquired over the course of 10 hours, to record the process of MPLs traveling through mucus and being internalized by cells. In certain systems, >70% of MPLs may be internalized by the intended cell type, and <5% by the unintended cell types. For example, fibroblast-targeting MPLs, when added to the fibroblast culture, are expected to be mostly ingested by fibroblasts, and only negligible amount, when added to the macrophage culture, should be detected within cells. Second, MPLs targeting different cell types are added, one type at a time, to the co-culture consisting of neutrophils, macrophages, fibroblasts and epithelial cells, with each cell type labeled with distinct fluorescent proteins.3D timelapse images are acquired over the course of 10 hours. In certain systems >70% of MPLs may be internalized by the intended cell type, and <5% by the unintended cell types. The measurement results will inform us at which viscosity MPLs specifically target and enter the intended cells. Optimization in MPL composition and synthesis protocols can be pursued to achieve optimal efficiency and specificity. [000321] Distribution and cytotoxicity: The single cell-type cultures are stained with viability dyes at the end of 10-hour imaging experiment. The number of total live and dead cells, with and without fluorescently-labeled MPLs are tabulated. The MPL distribution are calculated as the ratiobetween the per- centage of live cells containing MPLs to the percentage of the total live cells. The cytotoxicity are calculated as the ratio between the percentage of dead cells containing MPLs to the percentage of total cells (live and dead) containing MPLs. Cultures incubated with plain liposome serve as control. The percentage of the dead cells in the control group are compared to the calculated value of MPL cytotoxicity. If these two values are not significantly different, the cytotoxicity are deemed negligible. In certain systems, >70% distribution and <5% cytotoxicity may be achieved. Optimization in MPL dosage can be pursued to produce desired outcomes. [000322] In vitro evaluation wound-healing efficacy and long-term effect [000323] Overview: The efficacy of MPLs in restoring wound repair functions at viscous mucus typical of COPD patient in the BAT model can be evaluated. The effects of MPLs on dynamic cell behaviors can be evaluated. To understand the short- and long-term effects of MPLs, gene expression of each cell type in BAT can be profiled at various time points over the course of the MPL treatment. The results will help us determine the optimal regimen to continuously target key factors contributing to inflammation and fibrosis. [000324] MPL treatment: BAT samples exposed to CSE and covered with synthetic mucus are be subjected to MPL treatment. First, the efficacy of single MPL type is tested, and then various combinations of different MPL types. MPLs at BAT surface are administered with an optimized dosage. Varying administration frequencies can be tested, such as: 6-, 12-, 18-, 24-hour intervals. The treated BAT samples will be imaged longitudinally to monitor wound healing. A group of BAT samples will also be processed at different time points for gene expression profiling. [000325] Wound healing: Timelapse imaging of treated BAT samples can be performed, and analyze number of cells, cell speed, and persistence in closing wound. The results acquired from different group of treatment frequencies, at different time points, can be plotted against time and compared to those from the un- treated controls. We will identify the effective treatment frequencies improving wound healing in viscous mucus. [000326] Long-term effect: Treated BAT samples will be fixed on Day 1,3,5..., until Day 21 over the course of 21-day culture. The fixed samples will be subjected to sectioning, laser capture microdissection. Gene expression profiling will be performed in the dissected neutrophils / macrophages, fibro- blasts and epithelial cells. The expression of genes related to viscosity-induced inflammation and fibrosis identified in Aim 1 will be quantified using both qPCR184 and targeted mass spectrometry. The results acquired from different group of treatmentfrequencies, at different time points, will be compared to those from the untreated controls. Combining the results, we will further narrow down the effective treatment frequencies for optimal wound healing and inflammation / fibrosis suppression. [000327] Safety and efficacy evaluation using a mouse model [000328] Overview: Mice with mucus hypersecretion and airway denudation will be treated with MPLs. In the treated and control mice, the vital signs will be monitored; the wound-healing rate will be measured. [000329] Mouse model: The transgenic mouse model will be used, in which IL13 overexpression can be induced by doxycycline. The IL13-overexpressed mice, once treated with naphthalene, exhibit mucus hypersecretion, emphysema and airway denudation characteristic of muco-obstructive lung diseases. The optimized dosage and administration intervals are used first and adjusted if needed. The treatment starts on the 7th day post-IL13 induction and naphthalene administration and continues for 30 days. MPLs will be administered to the airway using a nebulizer. Mice treated using MPLs with empty cargos will be the control group. [000330] Safety evaluation: During the treatment, mice are closely monitored for distress using standard IACUC metrics. Treated mice are sacrificed every 3 days over 30 days. Airway tissues are dissected and processed for histological inspections. In particular, the area of airway tissue exhibit massive necrosis and the number of cells with positive staining of activated caspase 3, a marker for cell death, are quantified. [000331] Efficacy evaluation: Using an airway catheter, bronchoalveolar lavage (BAL) cell count differential can be performed to evaluate airway inflammation every 3 days following an established protocol. ELISA-based cytokine assays can be conducted in collected BAL fluids using an inflammatory cytokine panel. In addition, the tissue sections obtained can be used for MPL efficacy assessment. The total area of absent epithelium (wounds) and that of fibrotic tissues with overabundant collagen staining, can be quantified. The airspace morphometry for airspace dimension will also be performed by analyzing the histological images, to gauge the exacerbation of the muco- obstruction. [000332] Statistical Analysis and Expected Outcome [000333] Statistical Analysis: At least 30 tests per experimental condition in each sub aim are performed. To examine the effect of various viscosities and time points, results from different groups are analyzed using ANOVA followed by Tukey post-hoc tests. The values measured in differentgroups are deemed significantly different with p<0.01. At least 12 mice per group will be tested. A sample size of 12 per group is chosen to distinguish a 30% reduction in inflammation or fibrosis compared to the control with 89% power. [000334] Example 4: Preparation of drug-encapsulated mucus-penetrating liposomes [000335] We prepare drug-encapsulated mucus-penetrating liposomes (MPLs) These include encapsulating wound healing-improving drugs in mucus-penetrating liposomes (MPLs)1–3. An example of the protocol is shown in FIG.18. [000336] MPLs are surface-modified liposome capable of escaping the adhesive and steric interactions with mucin molecules, and penetrating the mucus rapidly. MPLs deliver the payload to the tissue underneath the mucus. This mucus penetrating capacity is achieved by grafting extremely dense layers of low-molecular-weight (<5 kDa) polyethylene glycol (PEG) onto the liposome surface. MPLs can penetrate viscous mucus nearly as rapidly as they diffuse in water4. MPLs will be synthesized using PEG conjugated with antibody targeting cell-type markers of fibroblasts (FAP),neutrophils / macrophages (CD11), and epithelial cells (E-cadherin), cell-type specificity. E-cadherin, localized at the lateral side of epithelial cells, is exposed to antibody conjugated to MPLs only at the wound site. By mixing drugs with lipids during synthesis, drugs can be encapsulated in MPLs5. Drug-carrying, mucus-penetrating vehicles can be utilized. Established protocols to prepare MPLs can be utilized containing one or more therapeutic agents including agents disclosed herein with appropriate cell-targeting antibody on the surface. For example, Trichostatin A-encapsulated MPLs with anti-FAP antibody on the liposome surface is a preferred composition. [000337] References for Example 4 1. Schneider, C. S., Xu, Q., Boylan, N. J., Chisholm, J., Tang, B. C., Schuster, B. S., Henning, A., Ensign, L. M., Lee, E., Adstamongkonkul, P., Simons, B. W., Wang, S.-Y. S., Gong, X., Yu, T., Boyle, M. P., Suk, J. S. & Hanes, J. Nanoparticles that do not adhere to mucus provide uniform and long-lasting drug delivery to airways following inhalation. Sci. Adv.3, (2017). 2. 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). 3. Yu, T., Chan, K. W. Y., Anonuevo, A., Song, X., Schuster, B. S., Chattopadhyay, S., Xu, Q., Oskolkov, N., Patel, H., Ensign, L. M., van Zjil, P. C. M., McMahon, M. T. & Hanes, J. Liposome-based mucus-penetrating particles (MPP) for mucosal theranostics: Demonstration of diamagnetic chemical exchange saturation transfer (diaCEST) magnetic resonance imaging (MRI). Nanomedicine Nanotechnology, Biol. Med.11, 401–405 (2015). 4. Popov, A. Mucus-Penetrating Particles and the Role of Ocular Mucus as a Barrier to Micro- and Nanosuspensions. J. Ocul. Pharmacol. Ther.36, 366–375 (2020).5. Sivadasan, D., Sultan, M. H., Madkhali, O. A., Alsabei, S. H. & Alessa, A. A. Stealth Liposomes (PEGylated) Containing an Anticancer Drug Camptothecin: In Vitro Characterization and In Vivo Pharmacokinetic and Tissue Distribution Study. Molecules 27, 1086 (2022). 6. Lai, S. K., O’Hanlon, D. E., Harrold, S., Man, S. T., Wang, Y.-Y., Cone, R. & Hanes, J. Rapid transport of large polymeric nanoparticles in fresh undiluted human mucus. Proc. Natl. Acad. Sci.104, 1482–1487 (2007). 7. Liu, M., Zhang, J., Shan, W. & Huang, Y. Developments of mucus penetrating nanoparticles. Asian J. Pharm. Sci.10, 275–282 (2015). 8. Ozpolat, B., Sood, A. K. & Lopez-Berestein, G. Liposomal siRNA nanocarriers for cancer therapy. Adv. Drug Deliv. Rev.66, 110–116 (2014). 9. Liu, P., Chen, G. & Zhang, J. A Review of Liposomes as a Drug Delivery System: Current Status of Approved Products, Regulatory Environments, and Future Perspectives. Molecules 27, 1372 (2022). 10. Guimarães, D., Cavaco-Paulo, A. & Nogueira, E. Design of liposomes as drug delivery system for therapeutic applications. Int. J. Pharm.601, 120571 (2021). 11. Alp, G. & Aydogan, N. Lipid-based mucus penetrating nanoparticles and their biophysical interactions with pulmonary mucus layer. Eur. J. Pharm. Biopharm.149, 45–57 (2020). OTHER EMBODIMENTS [000338] From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. [000339] All citations to sequences, patents and publications in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. By their citation of various references in this document, Applicants do not admit any particular reference is “prior art” to their disclosure.

Claims

What is claimed:

1. A method of treating a wound, injury, disease or disorder associated with an increase in viscosity of extracellular fluid (ECF) comprises: administering to a subject in need of treatment, a composition comprising a therapeutically effective amount of one or more agents, wherein the agents inhibit fibroblast cell motility, immune cell motility, induce transient epithelial-mesenchymal transition (EMT) or combinations thereof.

2. The method of claim 1, wherein the one or more agents comprise small molecule compounds, antisense reagents, siRNA reagents, antibodies, enzymes, peptides, organic or inorganic molecules, natural or synthetic compounds.

3. The method of claims 1 or 2, wherein the one or more agents modulate cellular ruffling.

4. The method of claim 3, wherein the one or more agents modulating cellular ruffling decrease total cofilin concentration in a cell as compared to a normal control.

5. The method of claim 3, wherein the one or more agents modulating cellular ruffling increase activated cofilin in a cell as compared to a normal control.

6. The method of claim 5, wherein the activated cofilin is increased by administration of a LIM kinase (LIMK) inhibitor.

7. The method of any one of claims 3 through 6, wherein the one or more agents modulating cellular ruffling inhibit one or more formin family members.

8. The method of claim 7, wherein a formin family member comprises Dia1.

9. The method of claim any one of claims 3 through 8, wherein the one or more agents modulating cellular ruffling comprise inhibitors of YAP nuclear translocation.

10. The method of claim 9, wherein an inhibitor of YAP nuclear translocation comprises YAP / TAZ inhibitor-1, histone deacetylases (HDACs) inhibitors, YAP-binding molecules, quinolinols, dopamine receptor D1 (DRD1) agonists or combinations thereof.

11. The method of claims 1 or 2, wherein the one or more agents induce re-epithelization, inhibit mucus triggered fibroblast activation, attenuate immune cell inflammatory responses or combinations thereof.

12. The method of claims 1 or 2, wherein the one or more agents modulate intracellular integrins, focal adhesion formation, actin polymerization or combinations thereof.

13. The method of claim 12, wherein modulators of integrins comprise: α5β1 integrin agonist-1 (C24H26FN5O9), αvβ1 integrin-IN-1 TFA (C28H35F3N6O8S), integrin-IN-2 (C27H3ON4O3),,integrin modulator 1 (C13H14N2O4),.

14. The method of any one of claims 1-12, wherein the one or more agents are functionalized for (i) mucus penetration, (ii) cell type-specific targeting, (iii) molecules treating epithelial cells, fibroblasts, or immune cells.

15. The method of any one of claims 1-3, wherein the one or more agents are encapsulated in a liposome, a microparticle or nanoparticle.

16. The method of claim 4, wherein the liposomes, microparticles or nanoparticles comprise one or more binding domains which specifically bind to one or more target cells.

17. The method of claim 5, wherein the one or more binding domains comprise antibodies, antibody fragments, Fab, Fab’, F(ab’)2fragments, Fv fragments, single chain antibodies, humanized antibodies and antibody fragments, camelized antibodies and antibody fragments, antibody mimetics, DARPins, affibody molecules, affilins, affitins, anticalins, avimers, fynomers, Kunitz domain peptides, monobodies, diabodies, tribodies, peptoids, aptamers, receptor fragments, ligands, oligosaccharides or combinations thereof.

18. The method of any one of claims 1 through 17, wherein the disease or disorder associated with an increase in viscosity of ECF comprise: cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), tumor microenvironments, lung inflammation, asthma, autoimmune diseases or combinations thereof.

19. The method of any one of claims 1 through 17 wherein the disorder or disorder is an ulcer, inflammatory bowel disease (IBD) or and mucinous cancer.

20. The method of any one of claims 1 through 19, wherein extracellular fluids (ECFs) comprise mucus, interstitial fluids, saliva, cerebrospinal fluids, synovial fluids, pleural fluids, pericardial fluids, peritoneal fluids, plasma or combinations thereof.

21. The method of any one of claims 1 through 20, further comprising combination therapies comprising administering an effective amount of one or more secondary therapeutic agents.

22. The method of claim 21, wherein the one or more secondary therapeutic agents comprise anti-inflammatory agents, cytokines, modulators of checkpoint inhibitors, ligands of checkpoint inhibitors, an agonist, an antagonist, an antibody, an antigen-binding fragment, a polynucleotide, a peptide or combinations thereof.

23. A method of identifying candidate therapeutic agents which modulate increase in viscosity of extracellular fluid (ECF) comprises: incubating a cell in media having varying viscosities, contacting the cell with one or more agents, assaying cellular responses to the one or more agents, and identifying a candidate therapeutic agent.

24. The method of claim 24, wherein the cell is an immune cell, an epithelial cell, a fibroblast, or combinations thereof.

25. The method of claim 23 or 24, wherein the cellular responses comprise: ruffling of lamellipodia, spreading, focal adhesion formation, migration, or combinations thereof.

26. The method of claim any one of claims 23 through 25, wherein the method is a high- throughput screening method.

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