WO2025199463A1 - Pectin films and manufacture thereof - Google Patents

Abstract

The present disclosure describes a method of preparing a bioadhesive, pectin- based polymer film. The method includes mixing high-methoxyl pectin (HMP) and water to form an HMP putty ball; hydrating a surface of the HMP putty ball; pressing the HMP putty ball into an HMP film of a substantially uniform thickness; re- hydrating the HMP film; and de-watering the re-hydrated HMP film to a point of at least partial gel-to-glass phase transition. Methods of treating a wound of a subject in need thereof using the bioadhesive, pectin-based polymer film are also described.

Description

PECTIN FILMS AND MANUFACTURE THEREOF

CLAIM OF PRIORITY

This application claims the benefit of U.S. Provisional Application Serial No. 63/568,235, filed on March 21, 2024. The entire content of the foregoing is incorporated herein by reference.

TECHNICAL FIELD

The present disclosure describes methods of preparing or manufacturing bioadhesive polymer compositions. In addition, the present disclosure describes bioadhesive polymer compositions including one or more films comprising a polymer comprising high methoxyl pectin (HMP) and water. In some embodiments, tannic acid is optionally added to the bioadhesive polymer compositions. The disclosure also describes methods of treating a wound in a subject in need thereof with these films.

BACKGROUND

The mesothelium is a membrane composed of simple squamous epithelial cells of mesodermal origin, which forms the lining of several body cavities: the pleura (pleural cavity around the lungs), peritoneum (abdominopelvic cavity including the mesentery, omentum, falciform ligament, and the perimetrium), and pericardium (fluid-filled sac surrounding the heart). Injury to the mesothelium creates a problem, not simply because of the compromised barrier function, but because the movement and forces associated with visceral organs (e.g., lung, bowel, and heart) can significantly compromise mesothelial healing. The slippery or non-adhesive surface of the mesothelium makes the use of traditional adhesives or sealants difficult. Thus, improvements in the manufacture and design of bioadhesives are continually sought.

SUMMARY

Certain aspects of the present disclosure are directed to methods of manufacturing or preparing bioadhesive, pectin-based polymer compositions comprising any feature described herein, either individually or in combination with any feature, in any configuration. Certain aspects of the present disclosure are directed to a method of preparing a bioadhesive, pectin-based polymer fdm, the method comprising: mixing high- methoxyl pectin (HMP) and water to form an HMP putty ball; hydrating a surface of the HMP putt}' ball; pressing the HMP putty ball into an HMP film of a substantially uniform thickness; re-hydrating the HMP film; and de-watering the re-hydrated HMP film to a point of at least partial gel-to-glass phase transition to form the bioadhesive, pectin-based polymer film.

In some embodiments, the HMP putt ' ball is formed with HMP present at an initial concentration of about 29% (w/w) to about 31% (w/w).

In some embodiments, the HMP is in a powder form when mixed with water to form the HMP putty ball.

In some embodiments, mixing the HMP and water comprises mixing the HMP and water using a high-speed bladeless mixer.

In some embodiments, mixing the HMP and water comprises subjecting the HMP and water to combined, opposing centrifugal forces generated by a mixer.

In some embodiments, hydrating the surface of the HMP putty ball comprises contacting the HMP putty ball with a hydrating medium.

In some embodiments, the HMP putt}' ball is contacted with the hydrating medium by placing the HMP putty ball within a container comprising the hydrating medium.

In some embodiments, the method further comprises swirling the HMP putt}' ball within the container until the HMP putty ball is evenly coated with the hydrating medium.

In some embodiments, the method further comprises placing the HMP putty ball on a substrate prior to pressing the HMP putty ball.

In some embodiments, pressing the HMP putty ball further comprises placing the HMP putt}' ball in a mold and using a hydraulic press to apply pressure to the mold, thereby pressing the HMP putty ball into the HMP film.

In some embodiments, the mold comprises two flat faces.

In some embodiments, the method further comprises pressing a frame into the HMP film prior to re-hydrating the HMP film. In some embodiments, re-hydrating the HMP film comprises adding a rehydrating medium into the frame and contacting the HMP film with the re-hydrating medium.

In some embodiments, de-watering the re-hydrated HMP film further comprises positioning the re-hydrated HMP film in a controlled environment until an equilibrium moisture content in the HMP film of about 20% is achieved.

In some embodiments, de-watering the re-hydrated HMP film further comprises using a desiccant.

In some embodiments, de-watering the re-hydrated HMP film further comprises de-watering the re-hydrated HMP film at an ambient condition.

In some embodiments, the bioadhesive, pectin-based polymer film has a thickness of about 120 pm to about 160 pm.

In some embodiments, the bioadhesive, pectin-based polymer film has a moisture content of about 14% to about 20%.

In some embodiments, the bioadhesive, pectin-based polymer film has a burst force of about 50 newtons (N) to about 110 N, and an extensibility of about 1 mm to about 4 mm.

Certain aspects of the present disclosure are directed to a method of sealing a wound in a tissue of a subject in need thereof, the method comprising: providing a bioadhesive pectin-based polymer film comprising: a high methoxyl pectin (HMP) and water, prepared by any of the methods disclosed herein; contacting the wound with the film; and applying pressure to the film, thereby sealing the wound in the tissue of the subject.

The terms “subject” or “patient” as used herein refer to any mammal (e.g., a human or a veterinary subject, e.g., a dog, cat, horse, cow, goat, sheep, mouse, rat, or rabbit) to which a composition or method of the present disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. The subject may seek or need treatment, require treatment, is receiving treatment, will receive treatment, or is under care by a trained professional for a particular disease or condition.

As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. As used herein, the term “active agent” is any molecule which is encapsulated, conjugated, fused, dispersed, embedded, mixed, or otherwise affixed to any of the film compositions described herein and is useful for a disease therapy.

As used herein, the expression “pharmaceutically acceptable” applies to a composition which contains composition ingredients that are compatible with other ingredients of the composition as well as physiologically acceptable to the recipient (e.g., a mammal such as a human) without the resulting production of excessive undesirable and unacceptable physiological effects or a deleterious impact on the mammal being administered the pharmaceutical composition. In some embodiments, a composition for use comprises one or more carriers, useful excipients, and/or diluents.

As used herein, the term “biodegradable” refers to a substance which may be broken down by microorganisms, or which spontaneously breaks down over a relatively short time (within about 14 days to about 6 months) when exposed to environmental conditions commonly found in a physiological environment. For example, the compositions described herein may be degraded by enzymes which are present in the body (e g., the mesothelial environment) or by gradual hydrolysis.

As used herein, the term “bioadhesive” refers to a composition that can securely bind to living tissue.

As used herein, the term “biocompatible” means a composition that is physiologically acceptable to a living tissue and organism.

As used herein, a “pectin” is any one of a family of galacturonic acid-rich polysaccharides including homogalacturonan, rhamnogalacturonan I, and the substituted galacturonans rhamnogalacturonan II (RG-II) and xylogalacturonan (XGA), as described in Mohnen, “Pectin Structure and Biosynthesis,” Current Opinions in Plant Biology, 11:266-277, 2008. High methoxyl pectins and amidated pectins are variations of the pectin family. As used herein, the terms “high-methoxyl pectin,” “high-methyl pectin,” and high methyl ester-pectin” are used interchangeably.

As used herein, the term “substantially uniform thickness” means a thickness that is largely or approximately consistent across the specified area, with minor variations that do not significantly affect the overall function or performance of the pectin film. For example, a substantially uniform thickness is a thickness that varies by no more than ±30% across the area of the pectin film. Ranges may be expressed herein as from “about” one particular value and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. The use of the term “about,” as used herein, refers to an amount that is near the stated amount by about 10% including increments therein. For example, “about” can mean a range including the particular value and ranging from 10% below that particular value and spanning to 10% above that particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

Where values are described in the present disclosure in terms of ranges, endpoints are included. Furthermore, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.

Various embodiments of the features of this disclosure are described herein. However, it should be understood that such embodiments are provided merely by way of example, and numerous variations, changes, and substitutions can occur according to those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of this disclosure.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

Some embodiments of the disclosure are described herein with reference to the accompanying figures. The description and figures make apparent to a person having ordinary skill in the art how some embodiments of the disclosure may be practiced. The figures are for the purpose of illustrative discussion and no attempt is made to show structural details of an embodiment in more detail than is necessary for a fundamental understanding of the teachings of the disclosure. Any figures herein are not shown to scale. Where dimensions are given in the text or figures, these dimensions are merely exemplary and do not limit the scope or spirit of the disclosed invention.

FIGs. 1A-1C are tables detailing an example method of manufacturing bioadhesive films. FIG. 1A is a table detailing the HMP ball preparation steps. FIG. IB is a table detailing an HMP ball preparation step and the rehydration steps. FIG. 1C is a table detailing the de-watering steps.

FIG. 2 is a photographic image of an example bioadhesive film.

FIG. 3 is a photographic image showing example frames resting on bioadhesive films placed on substrates.

FIG. 4 is a table showing example performance assessments and acceptance criteria of the bioadhesive films.

FIGs. 5A and 5B are graphs showing there was no significant effect of electron beam sterilization on the burst force (FIG. 5A) and extensibility (FIG. 5B) properties of the bioadhesive films.

FIG. 6 shows four photographic images of ex- vivo testing of the bioadhesive film on porcine lung tissue demonstrating immediate and sustained pleural adhesion.

FIG. 7 is a flowchart showing an example method of preparation HMP films by using film casting and drying.

DETAILED DESCRIPTION

The compositions described herein include bioadhesive polymer (e.g., pectin- based) film compositions. In some examples, the compositions described herein are used for sealing a wound (e.g., a wound in a visceral organ). Some embodiments of the fdm compositions and methods of manufacturing described herein may provide one or more of the following advantages.

Certain embodiments of the present disclosure include methods of manufacturing the bioadhesive, pectin-based polymer film compositions. Cunent methods of manufacturing polymer films often involve lengthy drying times and yield polymer films having thickness irregularities. Furthermore, attempts at accelerating the curing process with physical processes such as heat and vacuum tend to create irregularities including bubbles, warping and variable thickness. In some embodiments, the methods disclosed provide a simple and efficient method that is standardized and repeatable, and yields polymer films with consistent physical characteristics such as thickness, moisture content, burst force, and extensibility.

Certain embodiments of the present disclosure include bioadhesive, pectin- based polymer film compositions, some of which are referred to herein as pectin films. Some embodiments described herein may provide bioadhesive polymer films that may have tunable degradability and controlled release of a drug loaded into the film. For example, in some embodiments, the degradability (e.g., facial erosion) of the bioadhesive polymer films described herein can be controlled by varying the amount of tannic acid in the film composition. For example, the degradation half-life of the films can be increased by increasing the amount of tannic acid in the film compositions. By controlling the degradation half-life of the films, the release of a loaded drug (e.g., a hydrophilic drug or a hydrophobic drug) can also be controlled. Thus, in some embodiments, the compositions and methods of the disclosure may provide a flexible sealant that can be optimized for various tissue types, tissue injuries, and delivery cargoes.

In addition, some embodiments descnbed herein may provide a bioabsorbable sealant. For example, in contrast to sutures, the bioadhesive polymer compositions described herein are bioabsorbable so there is no need for postoperative removal of any compositions.

The film compositions and methods of the present disclosure provide a wound sealant that is simple, efficient, and effective. For example, in some embodiments, the films described herein do not require the addition of curing agents (e.g., photocrosslinkers) and additional steps (e.g., exposure to light) to initiate adhesion onto a tissue or to seal a wound in a tissue. The bioadhesive, pectin-based films of the disclosure adhere to a tissue within seconds.

For example, the methods of the disclosure can require an application routine that is reminiscent of the application of pressure sensitive adhesives (e.g., Scotch® tape). In other words, adhesion of the bioadhesive polymer films described herein can develop in seconds. Thus, in some embodiments, the film compositions and methods of the disclosure provide a user (e.g., a clinician) with an efficient method to easily seal a wound in a tissue that requires no additional steps other than contacting the bioadhesive film(s) with the tissue of interest. For example, in some embodiments, the methods described herein do not require a user to mix various components. In some embodiments, the bioadhesive polymer compositions of the disclosure do not have a limited time window within which a user must apply the composition to a tissue before an undesirable change to the composition occurs (e.g., unwanted solidification, unwanted separation of components, or the like). Thus, some embodiments described herein may provide methods of sealing a wound that are simple and time-efficient.

In another example, in some embodiments, the films described herein can have sufficient adhesive strength to adhere to a tissue (e.g., a visceral tissue and/or an ocular tissue) for a prolonged penod of time without falling off. In some embodiments, the films of the disclosure, once adhered to an injured tissue, can have a pressure resistance sufficient to remain adhered to the injured tissue through cyclical fluctuations (e.g., increase and decrease of pressure in a tissue or organ due to inspiration or expiration). In some embodiments, the films of the disclosure can help prevent wound leakage after surgery. The bioadhesive polymer compositions, e.g., pectin tape, can be capable of limiting wound leakage despite wide ranges in pressures.

Furthermore, the bioadhesive polymer compositions described herein are biocompatible and safe. For example, the bioadhesive polymer compositions include HMP, which has been widely recognized as a harmless food additive in North America and Europe. In the United States, pectin is affirmed GRAS (Generally Recognized as Safe) as defined in the Code of Federal Regulations (21 C.F.R. §184.1588). Film Compositions

The present disclosure features bioadhesive polymer compositions that can include one or more fdms comprising high-methoxyl pectin (HMP) and water. In some embodiments, tannic acid is optionally added to the bioadhesive polymer compositions.

Pectins are a family of plant cell wall polysaccharides and/or glycan domains that consist mainly of esterified D-galacturonic acid residues in a-(l->4) chains. Pectins differ from typical pressure sensitive adhesives, as they do not bind to most non-biologic compounds. However, they selectively and strongly bind to the mesothelial glycocalyx, which may be likely the result of a mechanism of interdiffusion or interpenetration, e.g., the entanglement of branched chain polysaccharides based on chemical bonds and weak chemical interactions.

Pectins can vary in molecular weight, cross-linking density (determined by multi-angle laser light scattering), and chemical groups (e.g., hydroxyl, amine, sulfur and carboxyl groups). The polysaccharides that make up pectin are generally grouped into three major types: homogalacturonan (HG), rhamnogalacturonan I (RG-I), and the substituted galacturonan rhamnogalacturonan II (RG-II). Some plant cell walls also contain additional substituted galacturonans, known as apiogalacturonan (AGA) and xylogalacturonan (XGA).

Thus, pectins are often defined by their source, e.g., citrus pectin (which was used in the examples described herein). The most common categories of pectin vary with respect to amidation and methoxylation; however, all pectins appear to be biocompatible. When exposed to calcium, pectin forms egg box-like structures that facilitate the immobilization of substances within the gel structure.

The pectins in the film compositions described herein are preferably high- methoxyl pectins (HMP), which can be obtained commercially (e.g., from Cargill, Inc., Minneapolis, MN, USA). The proportion of galacturonic acid residues in the methyl ester form determines the degree of methoxylation or methyl esterification. The proportion of galacturonic acid residues found in pectin in the methyl ester form determines the degree of methoxylation. HMPs are defined herein as those pectins with a degree of methoxylation equal to or greater than about 50%; low-methoxyl pectins (LMPs) are defined herein as those pectins with a degree of methoxylation of less than about 50%. As used herein, the term “initial concentration of HMP” refers to the concentration of HMP at the time when HMP is dissolved in water during the mixing step to form the HMP putty ball, as shown in FIG. 1A. In some embodiments, the pectin putty ball has an HMP concentration of about 29% (w/w) to about 31% (w/w) (e.g., about 29% to about 30% or about 30% to about 31% ( w)). The steps of preparing the fdms of the disclosure are disclosed below.

The loss of water alone from a dispersed solution of HMP can lead to the initial polymerization of the pectin. This so-called “gel transition” is associated with a discrete change in the physical properties of the pectin from a viscous liquid to a soft and rubbery gel. The ongoing loss of water from the pectin gel leads to a second discrete step, so called “glass transition,” associated with a change in the physical properties of the pectin from soft and rubbery' to hard and brittle. The compositions described herein include a film in a gel phase or a glass phase. In some embodiments, when in a gel phase, the film is soft, pliable, flexible, rubbery, moldable, or any combination thereof. In some embodiments, when in a glass phase, the film is rigid and brittle. In some embodiments, the bioadhesive film is a moldable film.

As described above, some embodiments of the films of the disclosure include tannic acid. Tannic acid is a specific form of tannin, a type of polyphenol, having a weak acidity' due to the numerous phenol groups in its structure. Without being bound to any theory, it is believed that tannic acid is a potential pectin chain crosslinker. In some embodiments, the films disclosed herein have a controlled degradation rate that is dependent upon the amount of tannic acid present in the film composition. For example, in some embodiments, the degradation half-life of the films increases with an increasing amount of tannic acid present in the film composition. In some embodiments, the loading of tannic acid into the films of the disclosure exhibits a dose-dependent decrease in pectin erosion.

As used herein, the term “initial concentration of water” refers to the concentration of water at the time when HMP is dissolved in water during the mixing step to form the HMP putty ball, as shown in FIG. 1A. In some embodiments, the pectin putty ball has an initial concentration of water of about 71% (w/w) to about 69% w/w) (e.g., about 71% to about 70% or about 70% to about 69% (wAv)).

In some embodiments, the concentration of water prior to the mixing step and formation of the HMP putty ball can different (e.g., greater) than the initial concentration of water. For example, in some embodiments, the methods of preparing the film composition can include a dissolving step in a pre-HMP putty ball preparation stage. In some embodiments, the dissolving step comprises dissolving HMP (and optionally tannic acid) in water to induce the film composition to be in a viscous solution state. In some embodiments, during the pre-HMP putty ball preparation stage, at least about 18 mL of water are present in the composition when the composition is in the viscous solution state. In some examples, during the pre- HMP putty ball preparation stage, the water concentration is at least about 85% (w/w).

In some embodiments, the bioadhesive films include one or more active agents. In some embodiments, the films exhibit a controlled release of one or more active agents that is determined by the degradation rate of the film or the facial erosion rate of the film, once adhered to a tissue. In some embodiments, the degradation rate or the facial erosion rate is determined by the amount of tannic acid present in the films. For example, in some embodiments, the degradation rate of the film or the facial erosion rate decreases with an increased concentration of tannic acid in the film. In some embodiments, the films have a controlled release profile governed by diffusion. For example, bioadhesive films that are composed of HMP and water (excluding tannic acid) and loaded with an active agent demonstrate a controlled release profile of the active agent, which is governed by diffusion.

In some embodiments, the bioadhesive film has a degradation half-life or a facial erosion half-life ranging from about 20 hours (h) to about 200 h (e.g., about 20 h to about 50 h, about 20 h to about 75 h, about 20 h to about 80 h, about 20 h to about 100 h, about 20 h to about 150 h, about 20 h to about 200 h, about 50 h to about 75 h, about 50 h to about 80 h, about 50 h to about 100 h, about 50 h to about 150 h, about 50 h to about 200 h, about 75 h to about 80 h, about 75 h to about 100 h, about 75 h to about 150 h, about 75 h to about 200 h, about 75 h to about 100 h, about 75 h to about 150 h, about 75 h to about 200 h, about 80 h to about 100 h, about 80 h to about 150 h, about 80 h to about 200 h, about 100 h to about 150 h, about 100 h to about 200 h, or about 150 h to about 200 h.) In some embodiments, the bioadhesive film has a degradation half-life of about 20 h. In some embodiments, the bioadhesive film has a degradation half-life of about 69 h. In some embodiments, the bioadhesive film has a degradation half-life of about 79 h. In some embodiments, the bioadhesive film has a degradation half-life of about 99 h. In some embodiments, the bioadhesive film has a degradation half-life of about 154 h.

In some embodiments, the bioadhesive films include one or more active agents, which can include an antibiotic, a procoagulant, a growth factor, a cytokine, or any combinations thereof.

In some embodiments, the one or more active agents can include a procoagulant. Non-limiting examples of procoagulants include thrombin, Factor Vila, Factor IX, Factor XIII, platelets, Von Willebrand Factor, fibrinogen, calcium ions, desmopressin, anti-inhibitor coagulant complex, antihemophilic factor (Factor VIII), prothrombin complex concentrate (PCC), andexanet alfa, aminocaproic acid, aprotinin, phytonadione (vitamin KI), protamine sulfate, and idarucizumab.

In some embodiments, the one or more active agents can include a hydrophobic drug, a hydrophilic drug, or both. For example, the one or more active agents can include one or more anti-bacterial agents or anti-fungal agents. Nonlimiting examples of anti-bacterial agents and anti-fungal agents include ciprofloxacin, levofloxacin, doxycycline hy elate, ofloxacin, erythromycin, cefazolin, vancomycin, gentamycin, tobramycin, ceftazidime, gatifloxacin, amphotericin, voriconazole, natamycin, bacitracin, besifloxacin, moxifloxacin, and tobramycin.

In some embodiments, the one or more active agents can include one or more anti-inflammatory agents. Non-limiting examples of anti-inflammatory agents include corticosteroids, loteprednol etabonate, prednisolone acetate, dexamethasone, lifitegrast, cyclosporine, bromfenac, nepafenac, ketorolac, diclofenac, suprofen, flurbiprofen, aspirin, ibuprofen, ketoprofen, non-steroidal anti-inflammatory drugs, or any combination thereof.

In some embodiments, the one or more active agents can include a cytokine. Non-limiting examples of cytokines include interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin- 10 (IL-10), interferon-alpha (IFN-a), interferon-gamma (IFN-y), tumor necrosis factor-alpha (TNF-a), granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming growth factor-beta (TGF-0), interleukin-12 (IL-12), interleukin-4 (IL-4).

In some embodiments, the one or more active agents can include one or more growth factors, e.g., one or more of transforming growth factor alpha (TGF-a) and TGF-p, tumor necrosis factor-alpha (TNF-a), vascular endothelial growth factor (VEGF), leukemia inhibitory factor (LIF), interleukins such as IL-1 through IL-7, colony-stimulating factors such as macrophage colony-stimulating factor (m-CSF), granulocyte colony-stimulating factor (G-CSF), and granulocyte macrophage colony- stimulating factor (GM-CSF), fibroblast growth factor (FGF), epidermal growth factor (EGF), insulin-like growth factor, connective tissue growth factor (CTGF), hepatocyte growth factor (HGF), angiopoi etin- 1-4, and platelet-derived growth factor (PDGF).

In some embodiments, the one or more films comprise the active agents. In some embodiments, the one or more active agents can include one or more of heparin, tissue plasminogen activator (tPA). In some embodiments, the one or more active agents can include hormones, cytokines, osteogenic factors, chemotactic factors, proteins and peptides that contain an arginine-glycine-aspartate (“RGD”) motif, analgesics, anesthetics, a vasoconstrictor, a clotting factor, a chemotherapy agent, an immunotherapy agent, or any combination thereof. In some embodiments, the vasoconstrictor includes one or more of norepinephrine, epinephrine, phenylpropanolamine, dopamine, metaraminol, methoxamine, ephedrine, and propylhexedrine. In some embodiments, the clotting factor includes fibrillar collagen, thrombin, fibrin, or any combination thereof. In some embodiments, the films of the disclosure do not include carboxymethylcellulose (CMC).

In some embodiments, the adhesive force of the bioadhesive films of the disclosure ranges from about 1 newton (N) to about 5 N (e g., about 1 N to about 2 N, about 1 N to about 3 N, about 1 N to about 4 N, about 2 N to about 3 N, about 2 N to about 4 N, about 2 N to about 5 N, about 3 N to about 4 N, about 3 N to about 5 N, or about 4 N to about 5 N). In some embodiments, the adhesive force of the bioadhesive films of the disclosure is about 3 N.

In some embodiments, tannic acid has no significant effect on the burst strength of the bioadhesive films of the disclosure. In some embodiments, the burst strength of the bioadhesive films of the disclosure ranges from about 30 newton (N) to about 50 N (e.g., about 30 N to about 35 N, about 30 N to about 40 N, about 30 N to about 45 N, about 30 N to about 49 N, about 35 N to about 40 N, about 35 N to about 45 N, about 35 N to about 50 N, about 40 N to about 45 N, about 40 N to about 50 N, or about 45 N to about 50 N). In some embodiments, the burst strength of the bioadhesive films of the disclosure is about 40 N. In some embodiments, tannic acid has no significant effect on the extensibility of the bioadhesive films of the disclosure. In some embodiments, the extensibility of the bioadhesive films of the disclosure decreases with an increasing concentration of tannic acid in the films. In some embodiments, the extensibility of the bioadhesive films of the disclosure ranges from about 2.5 millimeters (mm) to about 4.5 mm (e.g., about 2.5 mm to about 3 mm, about 2.5 mm to about 3.5 mm, about 2.5 mm to about 4 mm, about 3 mm to about 3.5 mm, about 3 mm to about 4 mm, about 3 mm to about 4.5 mm, about 3 mm to about 5 mm, about 3.5 mm to about 4 mm, about 3.5 mm to about 4.5 mm, about 3.5 mm to about 5 mm, about 4 mm to about 4.5 mm, about 4 mm to about 5 mm, or about 4.5 mm to about 5 mm). In some embodiments, the extensibility of the bioadhesive films of the disclosure is about 3 mm.

In some embodiments, the bioadhesive polymer film has a thickness ranging from about 40 pm to about 200 pm (e.g., about 40 pm to about 50 pm, about 40 pm to about 60 pm, about 40 pm to about 70 pm, about 40 pm to about 80 pm, about 40 pm to about 90 pm, about 40 pm to about 100 pm, about 40 pm to about 110 pm, about 40 pm to about 120 pm, about 40 pm to about 130 pm, about 40 pm to about 140 pm, about 40 pm to about 150 pm, about 40 pm to about 160 pm, about 40 pm to about 170 pm, about 40 pm to about 180 pm, about 40 pm to about 190 pm, about 40 pm to about 199 pm, about 50 pm to about 60 pm, about 50 pm to about 70 pm, about 50 pm to about 80 pm, about 50 pm to about 90 pm, about 50 pm to about 100 pm, about 50 pm to about 110 pm, about 50 pm to about 120 pm, about 50 pm to about 130 pm, about 50 pm to about 140 pm, about 50 pm to about 150 pm, about 50 pm to about 160 pm, about 50 pm to about 170 pm, about 50 pm to about 180 pm, about 50 pm to about 190 pm, about 50 pm to about 200 pm, about 60 pm to about 70 pm, about 60 pm to about 80 pm, about 60 pm to about 90 pm, about 60 pm to about 100 pm, about 60 pm to about 110 pm, about 60 pm to about 120 pm, about 60 pm to about 130 pm, about 60 pm to about 140 pm, about 60 pm to about 150 pm, about 60 pm to about 160 pm, about 60 pm to about 170 pm, about 60 pm to about 180 pm, about 60 pm to about 190 pm, about 60 pm to about 200 pm, about 70 pm to about 80 pm, about 70 pm to about 90 pm, about 70 pm to about 100 pm, about 70 pm to about 110 pm, about 70 pm to about 120 pm, about 70 pm to about 130 pm, about 70 pm to about 140 pm, about 70 pm to about 150 pm, about 70 pm to about 160 pm, about 70 pm to about 170 pm, about 70 pm to about 180 pm, about 70 pm to about 190 un, about 70 pirn to about 200 pun, about 80 pun to about 90 pun, about 80 pun to about 100 pun, about 80 pun to about 110 pun, about 80 pun to about 120 pun, about 80 pun to about 130 pun, about 80 pun to about 140 pun, about 80 pun to about 150 pun, about 80 pun to about 160 pirn, about 80 pirn to about 170 pun, about 80 pun to about 180 pun, about 80 pun to about 190 pun, or about 80 pun to about 200 pun). In some embodiments, the bioadhesive polymer composition comprises a film having a thickness of about 40 pm to about 50 pm.

In some embodiments, the bioadhesive polymer composition further includes a pharmaceutically acceptable carrier. As used herein, the expression “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation and is compatible with administration to a subject, for example a human. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits. Examples of pharmaceutically acceptable carriers include, but are not limited to, a solvent or dispersing medium containing, for example, water, pH buffered solutions (e.g., phosphate buffered saline (PBS), HEPES, TES, MOPS, etc.), isotonic saline, Ringer’s solution, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), alginic acid, ethyl alcohol, and suitable mixtures thereof. In some embodiments, the pharmaceutically acceptable carrier can be a pH buffered solution (e g. PBS).

In some embodiments, the pharmaceutically acceptable carrier is a topical carrier. In some embodiments, the bioadhesive polymer film is formulated for topical use. In some embodiments, the film is topically administered to a tissue (e.g., an ocular tissue or a mesothelial tissue) of a patient. In some embodiments, the film can be applied to a tissue (e.g., an ocular tissue or a mesothelial tissue) to seal an injury (e.g., an ocular injury or an injury to a mesothelial tissue). Methods of Preparing the Film Compositions

Certain embodiments of the disclosure include methods of preparing or manufacturing a bioadhesive, pectin-based polymer film. In some embodiments, the methods include providing a film comprising a polymer comprising HMP, tannic acid, and water. For example, the film can include any of compositions described elsewhere herein. In some embodiments, the method of manufacturing the bioadhesive, pectin-based polymer film is a “putty press” method, as described in FIGs. 1A-1C. In some embodiments, the method of manufacturing the bioadhesive, pectin-based polymer film is a film casting method, as described in FIG. 7.

In some embodiments, the putty press method of manufacture may begin by preparing a pectin (e.g., HMP) putty ball. As used herein, the term “putty” refers to a soft, malleable, doughlike material that can be shaped into a spherical body or ball. In some embodiments, the pectin putty ball is created by subjecting the pectin powder and water to the combined, opposing centrifugal forces generated by a mixer, which ensures both components are evenly mixed. In some embodiments, the pectin putty ball is formed using a bladeless centrifugal mixer (e.g., FlackTek™ SpeedMixer™). In some embodiments, the pectin putty ball has an HMP concentration of about 29% to about 31% (e.g., about 29% to about 30% or about 30% to about 31%) pectin by weight. For example, in some embodiments, about 3.3 g of pectin and about 7.7 mL of sterile water can be mixed to achieve a target pectin concentration of about 29% w/w) to about 31% w/w) pectin. In some embodiments, the pectin powder and water are mixed at about 2000 revolutions per minute (RPM) for about 4 to about 5 minutes. In some embodiments, a pectin putty ball is formed after the mixing step.

Next, in some embodiments, the methods of manufacturing include a step where the pectin putty ball is pressed into a film. In some embodiments, the surface of the pectin putty ball is uniformly hydrated prior to pressing the pectin putty ball. In some embodiments, this pre-pressing hydration step advantageously reduces film irregularities (e.g., fracture lines, stress marks, cracks, and/or “splay” marks) that are visually apparent in the final film product, as shown in FIG. 1A. In some examples, to hydrate the surface of the pectin putty ball, sterile water is poured into an empty, sanitized, mixing cup until the bottom of the cup is covered. Then, the pectin putty ball is placed into the cup and gently swirled around until the surface of the ball is evenly coated with water. Next, in some embodiments, the hydrated pectin putt)' ball is placed onto a substrate. In some embodiments, the substrate is a polyethylene terephthalate glycol (PETG) substrate. In some embodiments, the substrate is a nonstick substrate.

In some embodiments, the pectin putty ball is gently covered with a plastic film (e.g., a poly-film) once it is placed on the substrate. In some embodiments, the step of covering the pectin putty ball with a plastic film acts as a barrier and enables the pectin film to be cleanly removed from the mold. In some embodiments, the methods of manufacturing do not require covering the pectin putty ball with a plastic film. In some embodiments, the pectin putty ball is then pressed into a film. In some embodiments, a hydraulic press is used to press the pectin putty ball into a film. In some embodiments, a vertical molder is used to press the pectin putty ball into a film. In some embodiments, a mold is used along with the vertical molder or the hydraulic press to press the pectin putty ball. In some embodiments, the mold has two blank, flat faces. In other words, both faces of the mold are unmachined, with no modifications to their surfaces. In some embodiments, the mold is a steel mold. In some embodiments, the mold faces are unmachined or unprocessed. In some embodiments, an interchangeable insert mold system is used in the methods described herein. In some embodiments, the interchangeable insert mold system includes a blank mold insert having two blank, flat faces, as described above, and a master frame that is configured to receive the blank mold insert. In some embodiments, the master frame is operably connected or is part of the vertical molder or hydraulic press. In some embodiments, when using the interchangeable insert mold system, the pressing step comprises placing the blank mold insert into the master frame, positioning the substrate with the pectin putt}' ball on one of the flat faces of the blank mold insert, and then closing the mold insert to compress the pectin putty ball, ensuring a uniform thickness. In some embodiments, the pressing step comprises positioning the substrate with the pectin putty ball on one of the flat faces of the mold and then closing the mold insert to compress the pectin putty ball, ensuring a uniform thickness. In some embodiments, the vertical molder is used to apply a force to close the blank mold insert or mold.

Next, in some embodiments, the plastic film is carefully removed from the pressed pectin putty. In some embodiments, the pressed pectin putty is then re- hydrated. To begin the re-hydration process, a 3D-printed frame is first pressed into the flattened or pressed putty, as shown in FIG. 3. In some embodiments, the frame is composed of a chemically-resistant material that can withstand repeated cleaning and sanitization cycles. In some embodiments, the frame is rectangular. In some embodiments, the shape of the frame is customizable and any suitable shape of suitable dimensions can be used to cut the pressed pectin film into a desired shape. In some embodiments, the frame is a die used to cut out the pectin film.

Next, in some embodiments, a re-hydration medium (e.g., water) is poured into the frame in order to re-hydrate the pressed pectin putty. In some examples, about 10 mL to about 11 mL of sterile water are then poured into the frame. In some embodiments, the total re-hydration water volume is proportional to the frame dimensions. For example, 10 mL to about 11 mL of water are used for a frame dimension of about 8.6 cm by about 10.6 cm. In another example, if a frame having dimensions less than about 9 cm by about 11 cm is used, then the volume of water is less than about 10 mL to about 11 mL. In some embodiments, the substrate, containing the pressed pectin film, frame, and water within the frame, is gently tilted back and forth until the water is evenly dispersed on the pressed pectin putty surface within the frame. Next, in some embodiments, after the water is applied to the pressed pectin putty surface, the pressed pectin putty is covered with plastic wrap and allowed to re-hydrate overnight.

Pectins can demonstrate striking and reversible changes in their physical properties in the presence of even trace amounts of water. The loss of water alone from a dispersed solution of HMP can lead to the initial polymerization of the pectin. This so-called “gel transition” is associated with a discrete change in the physical properties of the HMP from a viscous liquid to a soft and rubbery gel. The ongoing loss of water from the HMP gel leads to a second discrete step, so-called “glass transition,” associated with a change in the physical properties of the HMP from soft and rubbery to hard and brittle. Thus, the methods described herein included progressively de-watering the film until the film was in a gel phase or a glass phase, as described below.

In some embodiments, the de-watering process starts by uncovering the pressed pectin putty, for example, by removing the plastic wrap, and moving it to a laminar flow hood for accelerated de-watering at ambient cleanroom conditions. In some embodiments, the ambient cleanroom conditions include a temperature ranging from about 15 °C to about 25 °C, and a humidity ranging from about 0% to about 60%. In some embodiments, the pressed pectin putty is de-watered under these conditions until the gel-to-glass phase transition is reached. In some embodiments, this endpoint is determined visually but typically occurs from about three to about five hours after the plastic wrap has been removed.

Next, in some embodiments, the pressed pectin putty is de-watered in a controlled environment to a target 20% equilibrium moisture content. In some embodiments, the controlled environment comprises exposing the pressed pectin putty to a desiccant. In some embodiments, the desiccant is a silica-based desiccant. In some embodiments, the silica-based desiccant comprises indicating silica gel desiccant beads. In some embodiments, method includes calculating a specific amount of desiccant required to remove a calculated amount of water from the pressed pectin putty. In some embodiments, the appropriate mass of desiccant is calculated based on the mass of the pressed pectin putty and the absorption capacity of the desiccant. In other words, the required mass of silica desiccant beads is calculated based on the mass of water that needs to be removed and the absorbency of the silica desiccant beads, as described in FIG. 1C. Then, in some embodiments, the partially dehydrated pressed pectin film is placed in a sealed polyethylene bag with the calculated amount of desiccant until equilibrium moisture content is achieved. In some embodiments, the endpoint of this step is determined through a visual assessment of the silica desiccant beads, as the beads change color from orange to black when fully saturated with moisture.

In some embodiments, following the de-watering stage of the method of manufacturing, the pectin film is removed from the substrate. In some embodiments, the pectin films start to peel away from the substrate when ready to be removed. FIG. 2 shows an example of a pectin film after it has been removed from the substrate. In some embodiments, after the pectin film is removed from the substrate, the pectin film is placed in a sealed, labeled moisture barrier container (e.g., a pouch). In some embodiments, the finished pectin films are sterilized (e.g., via electron beam sterilization). Testing of the Film Compositions

In some embodiments, the finished pectin films are tested by visual inspection and film thickness testing. In some embodiments, the de-watered pectin film thickness is dependent on the pectin putty ball concentration, the pressed pectin putty thickness, the volume of the re-hydration medium (e.g., sterile water), and de-watering time. In some embodiments, the dimensions of the de-watered film are controlled by controlling the dimensions of the 3D-printed frame. In some examples, the film is designed to have a length of about 5 cm to about 11 cm (e.g., about 5 cm to about 8 cm, about 6 cm to about 8 cm, about 7 cm to about 8 cm, about 8 cm to about 9 cm, about 8 cm to about 10 cm, or about 8 cm to about 11 cm), a width of 7 cm to about 13 cm (e.g., about 7 cm to about 10 cm, about 8 cm to about 10 cm, about 9 cm to about 10 cm, about 10 cm to about 11 cm, about 10 cm to about 12 cm, about 10 cm to about 13 cm), and a thickness of about 120 pm to about 160 pm (e.g., about 120 pm to about 140 pm, about 130 pm to about 140 pm, about 140 pm to about 150 pm, or about 140 pm to about 160 pm). In some examples, the film has length of about 8 cm, a width of about 10 cm, and a thickness of about 140 pm. In some embodiments, the frames are intentionally oversized to account for shrinkage during de-watering.

In some embodiments, a visual inspection is used to confirm that the finished pectin films have no cracks, folds, excessive curling, and/or warping. In some embodiments, a visual inspection is used to confirm that the finished pectin films have no visible undissolved pectin and no embedded particulates having an area greater than 0.20 mm². In some embodiments, a visual inspection is used to confirm that the finished pectin films have no bubbles having an area greater than 1 mm². In some embodiments, a visual inspection is used to confirm that the finished pectin films have less than three bubbles per 10 mm radius. In some embodiments, a visual inspection is used to confirm that the finished pectin films have no overt tackiness and/or that the finished pectin films do not stick to gloves during handling.

In some embodiments, the pectin films undergo burst testing using an Instron® material testing machine with a custom fixture. In some embodiments, the pectin films fabricated using the methods of manufacturing of the disclosure exhibit a burst force ranging from about 50 newtons (N) to about 110 N (e.g., about 50 N to about 80 N, about 60 N to about 80 N, about 70 N to about 80 N, about 80 N to about 90 N, about 80 N to about 90 N, about 80 N to about 100 N, about 80 N to about 110 N). In some embodiments, the pectin films undergo extensibility testing using an Instron® material testing machine with a custom fixture. In some embodiments, the pectin films fabricated using the methods of manufacturing of the disclosure exhibit an extensibility ranging from about 1 mm to about 4 mm (e.g., about 1 mm to about 3 mm, about 2 mm to about 3 mm, about 3 mm to about 4 mm, or about 2 mm to about 4 mm). In some embodiments, there is no significant effect on burst force and extensibility of the pectin films by sterilization (e.g., electron bean sterilization).

In some embodiments, moisture analysis is conducted using a loss-in-weight moisture analyzer. In some embodiments, the finished pectin films have a moisture content ranging from about 14% to about 20% (e.g., about 14% to about 17%, about 15% to about 17%, about 16% to about 17%, about 17% to about 18%, about 17% to about 19%, or about 17% to about 20%).

In some embodiments, the finished pectin films exhibit excellent optical clarity. In some embodiments, the finished pectin films manufactured using the methods described herein have de-watered film thicknesses that are consistently within the target range. In some embodiments, the finished pectin films undergo in vitro adhesivity testing. In some embodiments, the finished pectin films manufactured using the methods described herein show better adhesion compared to pectin films produced via a film casting method.

Methods of Treatment

In some embodiments, the films of the disclosure may facilitate the sealing of a visceral organ (e.g., lung, liver, bowel, heart, or any combination thereol) wound of a subject upon application and adhesion. In some embodiments, the films of the disclosure may facilitate the sealing of a wound of a mesothelial tissue of a subject upon application and adhesion. In some embodiments, the films of the disclosure may facilitate the sealing of a wound of a tissue having a glycocalyx surface (e.g., lung, liver, bowel, heart, eye, or any combination thereof) upon application and adhesion. In some embodiments, the HMP chains of the compositions exhibit entanglement with the glycocalyx of a tissue upon contact, thereby resulting in adhesion of the compositions to the tissue.

In some embodiments, the films of the disclosure may facilitate the sealing and/or treating of an ocular injury in an eye of a subject upon application and adhesion. For example, the films of the disclosure can be contacted with an ocular surface, can subsequently adhere to the ocular surface, and can seal an ocular injury. In some embodiments, the methods of sealing and/or treating an ocular injury include providing a bioadhesive polymer composition that is a single film. In some embodiments, the methods of sealing and/or treating an ocular injury include providing a bioadhesive polymer composition that includes two or more films adhered to each other.

Mesothelial Tissue Injuries

In some embodiments, the bioadhesive, pectin-based polymer film binds to a mesothelial tissue. In some embodiments, the mesothelial tissue includes one or more mesothelial tissues from a visceral organ. In some embodiments, the visceral organ is an internal organ of the abdominal, thoracic, and pelvic cavities. Non-limiting examples of visceral organs include one or more of a lung, a heart, a pancreas, a liver, a gall bladder, a spleen, a kidney, a stomach, a colon, a small intestine, a large intestine, and a bladder.

In some embodiments, the methods of sealing a wound in an injured mesothelial tissue of a subject can include providing a bioadhesive film comprising a polymer comprising HMP and water or a bioadhesive film comprising a polymer comprising HMP, tannic acid, and water (e.g., any of the bioadhesive films of the disclosure) that is prepared by any of the methods of manufacturing disclosed herein. Next, the methods can include contacting the wound of the subject with the bioadhesive polymer film. In some embodiments, the methods include applying pressure to the bioadhesive polymer film once it comes in contact with the wound or mesothelial tissue. In some embodiments, the pressure applied to the bioadhesive polymer film is a light pressure (e.g., about the same amount of pressure applied when adhering a piece of Scotch® tape to a paper). In some embodiments, the methods do not require the step of applying pressure to the bioadhesive film once it comes in contact with the wound. In some embodiments, the pressure is applied for about 10 seconds (s) or less (e.g., about 1 s to about 2 s, about 1 s to about 3 s, about 1 s to about 4 s, about 1 s to about 5 s, about 1 s to about 6 s, about 1 s to about 7 s, about 1 s to about 8 s, about 1 s to about 9 s, about 1 s to about 10 s, or about 5 s to about 10 s).

In some embodiments, the bioadhesive film reaches 80% of maximal adhesion to the wound or mesothelial tissue within at least about 5 seconds of contact, thereby sealing the wound in the mesothelial tissue of the subject. In some embodiments, the bioadhesive film adheres to the wound of the subject with an adhesion strength of at least about 3 N. In some embodiments, the bioadhesive film remains localized over the incision, injury, and/or laceration to seal the wound and form a surface barrier. In some embodiments, the bioadhesive film is a biocompatible and adhesive sealant on the ocular surface.

Ocular Injuries

The present disclosure presents methods and film compositions for treating ocular injuries (e.g., ocular surface injuries) in an eye of a subject. In some embodiments, the ocular injury is a comeal injury. In some embodiments, the ocular injury is a comeal incision. In some embodiments, the ocular injury is an injury or trauma resulting from an ocular surgery. In some embodiments, the ocular surgery is cataract surgery. In some embodiments, the compositions of the disclosure are used in post-surgical care. For example, the compositions can be administered to a patient after an ocular surgery to deliver a therapeutic agent (e.g., an anti-inflammatory agent or an antibiotic) that can be prescribed to minimize recovery time, prevent and/or treat inflammation caused by the surgical procedure, prevent and/or treat an ocular infection caused by the surgical procedure, or any combination thereof.

Ocular surface injuries can include conjunctival laceration, comeal perforation, scleral perforation, incisions due to ocular surgery (e.g., cataract surgery) or any combination thereof. In some embodiments, the ocular surface injury is a comeal or scleral injury. Conjunctival laceration can occur following blunt or penetrating trauma. Conjunctival lacerations can be associated with chemosis and subconjunctival hemorrhage. In such cases, it is important to rule out underlying scleral perforation. The fundus should be examined for any retinal tear or intraocular foreign body. An ultrasound can be done for the posterior segment evaluation.

Comeal lacerations and perforations represent approximately 1 in 10 of ocular traumatic injuries presenting in an emergency medical setting. Comeal lacerations and perforations can include partial thickness lacerations and full thickness lacerations. In addition, adnexal injuries, scleral perforation, or a combination thereof can be involved with comeal laceration and perforations. The standard of care for a comeal perforation includes the removal of any contaminants in the wound area, repair of the tear, and maintenance of the watertight integrity of the ocular globe. Comeal perforation can also be associated with or caused by insertion of a foreign body. In some embodiments, the comeal injury is a comeal full-thickness laceration or a comeal full-thickness perforation. In some embodiments, the ocular surface injury is a full-thickness laceration or a full -thickness perforation. In some embodiments, the ocular surface injury is a full-thickness laceration or surgical incision or a fullthickness perforation. For example, the majority of ocular surgeries that require entry into the eye (e.g., cataract surgery) involve a full-thickness incision through the cornea or sclera. Current management protocols for full thickness lacerations including scleral wounds often require the use of sutures.

The compositions of the disclosure can be used to treat ocular incisions or cuts or injuries having a length of less than about 1 mm to about 10 mm. In some embodiments, the compositions of the present disclosure can be used in the closure of full-thickness ocular defects and lacerations and in controlled and long-term drug elution. In some embodiments, indications can include post-operative applications of the biomaterial for drug elution in addition of closure of comeal ulcers, defects and perforations caused by a wide array of insults. The compositions of the disclosure can be applied both under “normal” (e.g., in-the-office or operating room) settings, or under emergency “in-the-field” settings. Various providers, physicians, and, in select cases, physician assistants and paramedics (e.g., in the combat theater) can apply the compositions described herein to seal the eye and elute drug(s) to heal defects. The compositions described herein can circumvent many cases of transplants and patch grafts for comeal melts and defects.

EXAMPLES

Certain embodiments of the present disclosure are further described in the following examples, which do not limit the scope of any embodiments described in the claims.

Example 1: Synthesis of Pectin Films by a “Putty Press” Method

The following process steps are also reflected in FIGs. 1A-1C. The process began by preparing an HMP putty ball using a bladeless centrifugal mixer (e.g., FlackTek™ SpeedMixer™) with about 3.3 g of pectin and about 7.7 mL of sterile water for a target pectin concentration of about 29-31% pectin by weight. Mixing was performed at about 2000 revolutions per minute (RPM) for 4 minutes. The output was a pectin putty ball. This mixing modality achieved homogenous pectin solutions with parameter-tuning, thereby enabling a broad range of concentrations and physical states.

Next, the method included a putty ball pressing step. A hydraulic press, e.g., a vertical molder, and blank mold insert were employed to press the putty ball into a film. Sterile water was poured into an empty, sanitized, mixing cup until the bottom of the cup was covered. A pectin putty ball was then placed into the cup and gently swirled around until the surface of the ball was evenly coated with water. The hydrated ball was placed onto a polyethylene terephthalate glycol (PETG) substrate and gently covered with a polymer film. The substrate was then placed onto the mold face and the mold was closed, thereby compressing the pectin ball flat and achieving a uniform thickness.

Next, the polymer film was carefully removed from the pressed putty. The pressed putty was then re-hydrated by pressing a 3D-printed frame into the flattened or pressed putty. (FIG. 3) Approximately 10-11 mL of sterile water (depending on the frame dimensions) were then poured into the frame. The water was not self-leveling on the putty surface, so the substrate was gently tilted back and forth until the water was evenly dispersed within the frame. The pressed putty or film was then covered with plastic wrap and allowed to re-hydrate overnight.

Following overnight re-hydration, the pressed putty' or film was uncovered and moved to a laminar flow hood for accelerated de-watering at ambient cleanroom conditions. The hydrated film was cut to shape prior to de-watenng. The putty was allowed to de-water under these conditions until the gel-to-glass phase transition was reached. This endpoint was determined visually but typically occurs 3-5 hours after the plastic wrap has been removed.

Final de-watering to a target 20% moisture content was performed in a controlled environment. The appropriate mass of desiccant was calculated based on the mass of the pressed putty or film sample, and the absorption capacity of the silica desiccant beads. In other words, the required mass of silica desiccant beads was calculated based on the mass of water that needed to be removed and the absorbency of the silica desiccant beads, as described in FIG. 1C. Then, the partially dehydrated pressed pectin film was placed in a sealed polyethylene bag with the silica desiccant beads until equilibrium moisture content was achieved. This was determined through a visual assessment of the silica desiccant beads, as the beads change color from orange to black when fully saturated with moisture.

Following de-watering, the pectin film, as shown in FIG. 2, was placed in a sealed, labeled moisture barrier pouch.

As shown in FIG. 4, finished film testing included visual inspection and film thickness testing. The de-watered film thickness is believed to be dependent on the pectin putty ball concentration, the pressed thickness, the volume of re-hydration media (sterile water), and de-watering time. The dimensions of the de-watered film were controlled with the dimensions of the 3D-printed frame. In this example, the film was designed to have a length of 8 cm ± 3 mm, a width of 10 cm ± 3 mm, and a thickness of 140 ± 20 pm. The frames were intentionally oversized to account for shrinkage during de-watering. Burst testing was performed using an Instron® material testing machine with a custom fixture. Burst force values achieved following this process were typically on the order of 35 newtons (N). Moisture analysis was conducted using a loss in weight moisture analyzer. The films produced in this hybrid process exhibited excellent optical clarity although they contained more microbubbles than films produced in the film casting or film pouring processes studied. The de-watered film thicknesses were consistently within the target range, and in vitro adhesivity testing showed better adhesion compared with films produced via the pectin pressing process alone (assessed qualitatively). The putty press method increased thickness and biodurability while maintaining adhesivity .

Example 2: Synthesis of Pectin Films via a Film Casting Process

Alternative pectin film synthesis approaches are possible. For example, a low viscosity pectin solution was prepared and poured into 3D-printed frames at set volumes to achieve the desired de-watered film thickness. An example process of this type is shown in FIG. 7.

The film casting process involved two phases: the HMP solution preparation and the film casting and drying phase. In the first phase, HMP powder was added in increments to double-distilled water to obtain a concentration of about 3% weight per volume (w/v). During the addition of the HMP powder to the water, a high shear, rotor-stator mixer (L5M-A Laboratory Mixer; Silverson®, East Longmeadow, MA) was used at 10,000 RPM to dissolve the HMP powder. Next, the mixing was continued for about 15 to 20 minutes to obtain a translucent solution devoid of solid particulates. Then, the solution was allowed to sit for about 45 to 60 minutes, after which it was ready to be cast into molds (e.g., petri dishes). This step marked the conclusion of the first phase.

The second phase or the film casting and drying phase started by using about 4 mL of the HMP solution to cast films in petri dishes having a 50 mm diameter. The appropriate volume of the HMP solution was withdrawn with a syringe, added to the petri dish while ensuring uniform solution distribution, and any air bubbles were removed. Next, the cast films were dried at ambient temperature (e.g., about 15 °C to about 25 °C) and ambient humidity (e.g., about 0% to about 60%) for about 12 hours. A second film layer was then cast by adding the same volume of HMP solution on top of the already dried first layer in the petri dish. The layered films were dried at ambient temperature and humidity for about 24-36 hours, and the dried films were stored in covered petri dishes at ambient conditions until further use. The experiments performed suggested increased warping or curling of the films as the pour volume was increased.

Example 3 - Ex vivo Testing of HMP Films on Porcine Lung Tissue

The pectin-based polymer films from Example 1 were tested on a porcine lung tissue ex vivo for a bioadhesion study, as shown in FIG. 6. Briefly, the pectin film was applied to and contacted the porcine lung tissue. Adhesion strength reflected the applied force required for interface separation. Adhesion of the pectin film to the porcine lung tissue was observed within a few minutes. The pectin film did not adhere to gloves or surgical instruments during the experiment. Adhesion of the pectin film to the porcine lung tissue was maintained even after application of force (e.g., pulling the pectin film away from the porcine lung tissue).

It will be appreciated by those skilled in the art that while the disclosed subject matter is described above in connection with particular embodiments and examples, the invention is not necessarily so limited, and that numerous other embodiments, examples, uses, modifications and departures from the embodiments, examples and uses are intended to be encompassed by the claims attached hereto. Each reference cited herein is incorporated by reference in its entirety.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of preparing a bioadhesive, pectin-based polymer film, the method comprising: mixing high-methoxyl pectin (HMP) and water to form an HMP putty ball; hydrating a surface of the HMP putty ball; pressing the HMP putty ball into an HMP film of a substantially uniform thickness; re-hydrating the HMP film; and de-watering the re-hydrated HMP film to a point of at least partial gel-to-glass phase transition to form the bioadhesive, pectin-based polymer film.

2. The method of claim 1, wherein the HMP putty ball is formed with HMP present at an initial concentration of about 29% (w/w) to about 31% (w/w).

3. The method of any one of claims 1-2, wherein the HMP is in a powder form when mixed with water to form the HMP putty ball.

4. The method of any one of claims 1-3, wherein mixing the HMP and water comprises mixing the HMP and water using a high-speed bladeless mixer.

5. The method of any one of claims 1-4, wherein mixing the HMP and water comprises subjecting the HMP and water to combined, opposing centrifugal forces generated by a mixer.

6. The method of any one of claims 1-5, wherein hydrating the surface of the HMP putt}' ball comprises contacting the HMP putty ball with a hydrating medium.

7. The method of claim 6, wherein the HMP putty ball is contacted with the hydrating medium by placing the HMP putty ball within a container comprising the hydrating medium.

8. The method of claim 7, further comprising swirling the HMP putty ball within the container until the HMP putty ball is evenly coated with the hydrating medium.

9. The method of any one of claims 1-8, further comprising placing the HMP putty ball on a substrate prior to pressing the HMP putty ball.

10. The method of any one of claims 1-9, wherein pressing the HMP putty ball further comprises placing the HMP putty ball in a mold and using a hydraulic press to apply pressure to the mold, thereby pressing the HMP putty ball into the HMP film.

11. The method of claim 10, wherein the mold comprises two flat faces.

12. The method of any one of claims 1-11, further comprising pressing a frame into the HMP film prior to re-hydrating the HMP film.

13. The method of claim 12, wherein re-hydrating the HMP film comprises adding a re-hydrating medium into the frame and contacting the HMP film with the rehydrating medium.

14. The method of any one of claims 1-13, wherein de-watering the re-hydrated HMP film further comprises positioning the re-hydrated HMP film in a controlled environment until an equilibrium moisture content in the HMP film of about 20% is achieved.

15. The method of claim 14, wherein de-watering the re-hydrated HMP film further comprises using a desiccant.

16. The method of any one of claims 1-15, wherein de-watering the re-hydrated HMP film further comprises de-watering the re-hydrated HMP film at an ambient condition.

17. The method of any one of claims 1-16, wherein the bioadhesive, pectin-based polymer film has a thickness of about 120 pm to about 160 pm.

18. The method of any one of claims 1-17, wherein the bioadhesive, pectin-based polymer film has a moisture content of about 14% to about 20%.

19. The method of any one of claims 1-17, wherein the bioadhesive, pectin-based polymer film has a burst force of about 50 newtons (N) to about 110 N, and an extensibility of about 1 mm to about 4 mm.

20. A method of sealing a wound in a tissue of a subject in need thereof, the method comprising: providing a bioadhesive pectin-based polymer film comprising: a high methoxyl pectin (HMP) and water, prepared by the method of any one of claims 1-19; contacting the wound with the film; and applying pressure to the film, thereby sealing the wound in the tissue of the subject.