Capsules for digestive tract sampling

Abstract

A passive device for non-invasive gastrointestinal sampling is disclosed. In some embodiments, the capsule device includes a capsule housing having a sampling opening and a biodegradable coating that seals the sampling opening. The biodegradable coating may be disposed within the sampling opening. The capsule contains an absorbent sampling hydrogel and a sealing member between the sampling hydrogel and the sampling opening. The capsule is ingested by a patient. When the capsule reaches a desired location in the gastrointestinal tract, the biodegradable coating dissolves to allow gastrointestinal fluids to enter the capsule and be absorbed by the sampling hydrogel. Absorption of the fluid causes the sampling hydrogel to expand, thereby pressing the sealing member against the sampling opening to seal the capsule. In some embodiments, the biodegradable coating may have multiple layers that may dissolve at different locations in the gastrointestinal tract to facilitate targeting a specific location, for example, the colon.

Description

Technical Field

[0001] The disclosed embodiments relate to capsules for gastrointestinal sampling and related methods of use.

Background Art

[0002] The development of new tools that can accurately sample target microbiota, proteins, chemicals, and other biomarkers throughout the gastrointestinal (GI) tract can facilitate more effective and accurate prediction and diagnosis of diseases and disease progression.

[0003] Many studies have found that the gut microbiota plays an important role in the pathophysiology that governs human health. Many of these studies have identified the effects of the gut microbiota on human metabolism, nutrient uptake, the effectiveness of orally administered therapies, and the function of the immune and nervous systems. For example, some studies have found correlations between dysbiosis and various diseases, including diabetes, obesity, and metabolic syndrome, which affect approximately 30 million people in the United States. Similarly, recent insights into the possible ways in which gut bacteria can affect the development and maintenance of the nervous system suggest a link between gut microbiome composition and the regulation of neuropsychiatric disorders, including dysbiosis in anxiety, depression, and autism spectrum disorder.

[0004] Furthermore, many diseases are associated with the production of different biomarkers and mRNA transcripts within the GI tubules. For example, fecal calprotectin (fCal) has been shown to be the most accurate in detecting inflammatory bowel disease (IBD) and mucosal damage. While several studies have demonstrated an association between fCal levels and the degree of inflammation, calprotectin levels are highly dependent on several factors, including diet, water content in the stool sample, and disease location. Patients with ileal Crohn's disease (CD) may have large ulcers with low levels of fCal. This may suggest that stool analysis allows for a simplified assessment of potential IBD but may not indicate a possible disease location or condition. [Overview of the project]

[0005] In one embodiment, a device for passive sampling of the digestive tract comprises a capsule housing defining a cavity, a sampling opening formed within the capsule housing and providing fluid communication between the cavity and the outside of the capsule housing, and a sampling hydrogel located within the cavity. Upon exposure to a sample fluid, the sampling hydrogel is configured to absorb the sample fluid, expand within the cavity, and store the sample fluid for subsequent analysis. The device further includes a sealing member located between the sampling hydrogel and the sampling opening within the cavity. The expansion of the sampling hydrogel within the cavity presses the sealing member and engages with the sampling opening, thereby sealing the cavity. The device further includes a biodegradable coating covering the sampling opening. The biodegradable coating comprises multiple biodegradable coating layers. Decomposition of the multiple biodegradable coating layers exposes the sampling opening, allowing fluid flow into the cavity.

[0006] In another embodiment, a method for manufacturing a device for passive sampling of the digestive tract includes providing a capsule housing having a cavity and a sampling opening. The sampling opening provides fluid communication between the cavity and the outside of the capsule housing. The method further includes closing the sampling opening with a biodegradable coating comprising multiple biodegradable coating layers. Decomposition of the multiple biodegradable coating layers exposes the sampling opening, allowing fluid flow into the cavity.

[0007] In one embodiment, a device for passive sampling of the gastrointestinal tract comprises a capsule housing defining a cavity, a sampling opening formed within the capsule housing and providing fluid communication between the cavity and the outside of the capsule housing, and a sampling hydrogel located within the cavity. Upon exposure to a sample fluid, the sampling hydrogel is configured to absorb the sample fluid, expand within the cavity, and store the sample fluid for subsequent analysis. The device further includes a sealing member located between the sampling hydrogel and the sampling opening within the cavity. The expansion of the sampling hydrogel within the cavity presses the sealing member and engages with the sampling opening, thereby sealing the cavity. The device further includes a biodegradable coating disposed within the sampling opening. Decomposition of the biodegradable coating exposes the sampling opening, allowing fluid flow into the cavity.

[0008] The concepts described above and the additional concepts discussed below may be arranged in any preferred combination, and it should be understood that this disclosure is not limited in this respect. Furthermore, other advantages and novel features of this disclosure will become apparent from the following detailed description of various non-limiting embodiments when considered in relation to the accompanying drawings. [Brief explanation of the drawing]

[0009] The attached drawings are not intended to be drawn to scale. In the drawings, identical or nearly identical components illustrated in various figures may be represented by the same number. For clarity, not all components may be labeled in all drawings. [Figure 1] This is a schematic diagram showing capsules moving through a GI tube according to several embodiments. [Figure 2A] A drawing shows one embodiment of the capsule at a first position within the GI tube. [Figure 2B] Draw the capsule in Figure 2A at the second position inside the GI tube. [Figure 2C] Draw the capsule in Figure 2A at the third position inside the GI tube. [Figure 2D] Draw the capsule in Figure 2A at the fourth position inside the GI tube. [Figure 3] This is a partial exploded view of a capsule according to several embodiments. [Figure 4A] This is a schematic diagram showing the first step of a method for manufacturing a coating for a capsule according to several embodiments using a drop casting technique. [Figure 4B] This is a schematic diagram showing the second step of the method in Figure 4A. [Figure 4C] This is a schematic diagram showing the third step of the method in Figure 4A. [Figure 4D] This is a schematic diagram showing the fourth step of the method in Figure 4A. [Figure 4E] This is a schematic diagram showing the fifth step of the method in Figure 4A. [Figure 5] Draw an alternative embodiment of the capsule. [Figure 6A] This plot shows the solubility profile of Eudragit L100-55 at various pH levels. [Figure 6B] This plot shows the solubility profile of Eudragit L100 at various pH levels. [Figure 6C]A plot showing the dissolution profile of Eudragit S100 at various pH levels. [Figure 6D] A bar graph showing the percentage of biodegradable coating polymer dissolved after 2-hour exposure to various pH levels. [Figure 7A] A plot showing the dissolution profile of Eudragit EPO at various pH levels. [Figure 7B] A plot showing the dissolution profile of chitosan at various pH levels. [Figure 7C] A bar graph showing the percentage of biodegradable coating polymer dissolved after 1-hour exposure to various pH levels. [Figure 8] A bar graph showing the cytotoxicity of the biodegradable coating before and after NIR drying. [Figure 9A] A plot showing the spectroscopic results for enteric polymers in powder form. [Figure 9B] A plot showing the spectroscopic results for enteric polymer films. [Figure 10A] A plot showing the thermogravimetric analysis of various forms of Eudragit L100-55. [Figure 10B] A plot showing the thermogravimetric analysis of various forms of Eudragit EPO. [Figure 11A] A microscopic image of a double-layer biodegradable coating dried in the ambient environment. [Figure 11B] A microscopic image of the double-layer biodegradable coating in Figure 11A after 2-hour exposure to a pH of 1.2. [Figure 11C] A microscopic image of the double-layer biodegradable coating in Figure 11B after 6-hour exposure to a pH of 6.8. [Figure 11D] A microscopic image of the double-layer biodegradable coating in Figure 11C after 1-hour exposure to a pH of 5.5. [Figure 11E] A microscopic image of a double-layer biodegradable coating dried using NIR. [Figure 11F]This is a microscopic image of the bilayer biodegradable coating in Figure 11E after 2 hours of exposure to a pH of 1.2. [Figure 11G] This is a microscopic image of the bilayer biodegradable coating in Figure 11F after 6 hours of exposure to a pH of 6.8. [Figure 11H] This is a microscopic image of the bilayer biodegradable coating in Figure 11G after 1 hour of exposure to a pH of 5.5. [Figure 12A] This is an SEM image of a double-layer biodegradable coating dried in its surrounding environment. [Figure 12B] This is a SEM image of a bilayer biodegradable coating dried using NIR. [Figure 13A] This bar graph shows the microbiome composition of the GI tube and a comparison with the capsule administered to the first animal. [Figure 13B] This bar graph shows the microbiome composition of the GI tube and a comparison with the capsule administered to the second animal. [Modes for carrying out the invention]

[0010] At least some embodiments described herein relate to targeted sampling of microbial communities, proteins, chemicals, and other biomarkers across the entire GI tube.

[0011] Human microbiome sampling has become an essential aspect of understanding the mechanisms of microbiome-drug interactions and the extent to which these complex interactions can affect drug efficacy and bioavailability. Much of what is known about the structure and function of the human gut microbiome has been confirmed through ex vivo culture and / or sequencing of bacteria from fecal samples. However, since only a small fraction of gut bacteria is available and culturable from fecal samples, efforts have been made to develop tools that would enable direct sampling of microorganisms from the gastrointestinal (GI) tract. For example, colonoscopy and / or gastroscopy are currently used, but these methods are limited to sampling in specific sections of the entire gastrointestinal tract and are invasive approaches that can cause discomfort to patients and lead to decreased compliance. Other approaches have involved the use of smart functional capsules capable of collecting samples at various target locations within the gastrointestinal tract, and these methods may address some of the limitations associated with conventional colonoscopy and gastroscopy. Furthermore, capsule-based devices may improve patient comfort without the need for administration in the clinical setting. For example, PillCam® capsule endoscopy (CE) technology is used to collect images from hard-to-reach areas throughout the gastrointestinal tract to diagnose ambiguous gastrointestinal bleeding, tumors, Crohn's disease, angiogenesis imperfecta, celiac disease, and small intestine-related diseases such as polyposis. However, this technology does not have the ability to collect and store samples as they pass through the gastrointestinal tract.

[0012] Efforts to develop novel capsules for sampling the gut microbiome using different methods can be categorized into two main categories: active devices and passive devices. In active devices, the operating and sampling mechanism is often achieved by using an onboard battery that provides the energy necessary to operate various plungers, pistons, biopsy forceps, etc., that collect and store samples within the capsule. However, in such devices, the battery often occupies a large portion of the capsule's volume, which can limit the space available for sample storage. Furthermore, active devices typically have a high risk of failure and the possibility of leakage of corrosive electrolytes, which can cause severe corrosive damage and liquefaction necrosis. To avoid these and other drawbacks associated with active devices, various passive operating sampling mechanism approaches have been employed, thereby enabling capsules to be more compact and economically viable while having fewer safety-related issues. In such designs, the capsule moves through a GI tube at an average speed of 1-2 cm / min via peristaltic motion, and the sample is collected by a simple sample passive operation, such as capillary wicking or pressure difference force. However, the inventors have recognized and appreciated that there are many important design considerations that have not yet been addressed by existing passive devices. For example, some existing approaches rely on the assembly of capsules that require a well-sealed vacuum chamber inside the capsule, which significantly increases the complexity of the device. Other approaches have relied on retrieving the sampling device via a string extending upstream into the gastrointestinal tract, which can cause discomfort to the patient.

[0013] In some embodiments, a capsule is provided having an opening that forms an opening from the outside to the inside of the capsule. The opening may be covered with a pH-sensitive enteric polymer coating, which dissolves when it enters the basic environment of the small intestine. In some embodiments, a hydrogel that draws a fluid sample into the capsule is located inside the capsule. When the fluid enters the capsule, the hydrogel swells. An elastomer disc may be provided with the hydrogel. When the hydrogel swells, the elastomer disc may be pressed against the opening, sealing the capsule from the inside, thereby preventing any further fluid exchange.

[0014] The inventors recognize that in some cases, arranging a biodegradable coating on the outside of the capsule to cover the opening may require the capsule to have corners or collars near the opening in order to contain the liquid solution during application.

[0015] Therefore, the inventors understand that in some embodiments, it may be beneficial to place the biodegradable coating inside the opening rather than on the outside of the capsule. Placing the biodegradable coating inside the opening may allow the outside of the capsule to exhibit a wider range of geometric shapes, including smoother, more rounded shapes that are better suited to passing through a GI tube. For example, the capsule may be elongated elliptical in shape, with only a small opening formed at one end. The biodegradable coating may be placed only inside the opening, which allows the outside of the capsule to have only rounded features. Alternatively or in addition, in some embodiments, placing the biodegradable coating inside the opening may increase ease of manufacture. For example, in some embodiments, fabricating the coating layer by depositing the coating material within the accommodating volume of the opening may be easier than applying the coating material around the outer surface of a round capsule. In some embodiments, depositing the coating material within the accommodating volume of the opening may facilitate control of the coating layer thickness. This may be particularly beneficial when multiple coating layers are applied, as will be discussed below.

[0016] Another limitation of current designs is that they target only the small intestine. Biodegradable coatings must withstand degradation in acidic environments and survive in the stomach, but dissolve in basic environments to enable sampling in the basic environment of the small intestine. Because the large intestine is more acidic, a single pH-sensitive coating that can withstand both the acidic environment of the stomach and the basic environment of the small intestine, reach the colon intact, and dissolve in the colon has not been identified. This limitation of enteric-coated polymer coating materials hinders the development of similar passive sampling devices for the colon. As mentioned above, currently available techniques for colon sampling are generally invasive, expensive, time-consuming, and require skilled technicians. Therefore, there remains an unmet need for targeted passive sampling from the colon that is more user-friendly.

[0017] The inventors recognize that in some embodiments, it may be beneficial to have multiple layers of a biodegradable coating, each layer dissolving at a different pH level. Such an arrangement may allow the capsule to target different areas of the body for passive sampling. For example, to target passive sampling from the colon, the outer layer may dissolve in a basic environment, and the inner layer may dissolve in an acidic environment. The outer layer may survive through the stomach, protecting the inner layer from the acidic environment within the stomach. The outer layer may dissolve in the basic environment of the small intestine, thereby exposing the inner layer. The inner layer may survive in the small intestine and dissolve in the acidic environment of the colon, thereby allowing the passive sampling process to occur within the colon.

[0018] The inventors recognize that in some embodiments, it may be useful to use a drop casting process to form one or more biodegradable coatings. They understand that drop casting can be used to generate thicker layers in a shorter time while maintaining control and simplicity in the application process. For example, an enteric polymer may be dissolved in a liquid solvent to form a polymer solution. Droplets of the polymer solution, having a controlled or known size, may be deposited on a surface or into an opening with controlled momentum, rapidly creating a coating layer of known thickness. However, it should be understood that in other embodiments, other methods may be used to form biodegradable coatings.

[0019] The inventors also recognize that, in some cases, the biocompatibility of the coating may be impaired if the solvent is trapped within the polymer matrix when the solvent used to prepare the polymer solution is toxic or otherwise biocompatible. Additionally, the solvent may act as a plasticizer within the polymer matrix, thereby potentially reducing the rigidity of the coating layer and potentially increasing the likelihood of premature seal failure. Therefore, the inventors understand that, in some embodiments, it may be desirable to remove the solvent from the coating layer after deposition.

[0020] The inventors recognize that in some embodiments, it may be useful to use near-infrared (NIR) spectroscopy to dry polymer solutions and remove the solvent after deposition. In the NIR process, waves of known, controlled wavelengths are emitted in the sample material. While NIR has been conventionally used as a method for identifying unknown materials, the ability to control the emitted wavelengths allows for the selection of wavelengths that may have higher absorbance in the solvent than in the enteric polymer. Higher absorbance in the solvent may lead to evaporation of the solvent, while lower absorbance in the enteric polymer may result in little to no damage to the polymer film structure. For example, the NIR process may allow the solvent to evaporate from the polymer matrix in a relatively short time, thereby allowing the solvent to be removed before the polymer matrix is ​​damaged. However, it should be understood that in other embodiments, other methods may be used to remove the solvent, such as heat.

[0021] Additionally, in designs having multiple layers of biodegradable coatings, the ability of each layer to dissolve at a desired point within the GI tubule may depend on the separation or stratification of different biodegradable coatings into separate layers. When different biodegradable coatings are mixed together, the effect is similar to creating a single layer with a concentration gradient throughout. The result is that the overall coating may partially dissolve in different parts of the GI tubule, potentially impairing the ability of the capsule to specifically target a particular point within the GI tubule (e.g., the colon). To prevent mixing, each layer of the coating may be dried or cured after deposition and before any subsequent layers are deposited.

[0022] In view of the above, the inventors recognize that in some embodiments, it may be beneficial to apply NIR to the coating layer before applying subsequent layers. For example, NIR may be used to rapidly dry and remove the solvent from the first polymer coating layer after the first polymer coating layer has been drop-cast into the opening of the sampling capsule and before the second polymer coating layer has been drop-cast onto the first layer. NIR may then be used to rapidly dry and remove the solvent from the second layer, resulting in a biocompatible coating having multiple distinct layers that are not structurally impaired. However, it should be understood that in other embodiments, other methods may be used to achieve solidification, for example, heat may be used.

[0023] In some embodiments, the passive sampling device may include a capsule, which may be ingested by a patient and thereby travel through a GI tube. The capsule may include a capsule housing defining a cavity and an absorbent sampling hydrogel located within the cavity. The capsule housing may include a sampling opening that allows fluid from the digestive tract (e.g., fluid containing microorganisms or proteins such as calprotectin) to flow into the cavity and be absorbed thereby by the sampling hydrogel. Upon absorbing the fluid, the sampling hydrogel may expand within the cavity, pressing against and engaging a sealing member (e.g., a sealing membrane) located in the cavity between the opening and the hydrogel material, thereby sealing the opening and thereby restricting subsequent fluid from entering or leaving the capsule. Thus, the devices disclosed herein may utilize the sampling hydrogel as a medium for storing microbial samples within a capsule and as a means of providing a passive mechanical action for sealing the capsule after sampling is complete. In addition, the inventors have understood that after the capsule is sealed, the hydrated sampling hydrogel within the capsule may provide an ideal living environment with nutrients for the bacteria sampled before the capsule is retrieved. Furthermore, according to some embodiments, sealing the capsule can help protect the sample collected within the capsule from the harsh environment located in the gastrointestinal tract, thereby preserving the bacterial sample stored in the sampling hydrogel sample for subsequent analysis.

[0024] In some embodiments, the capsule may include a biodegradable coating, which comprises multiple biodegradable coating layers disposed within the sampling opening. For example, the biodegradable coating may be an enteric coating configured to dissolve at a desired target position along the length of the GI tube. In one embodiment, an outer biodegradable coating layer and an inner biodegradable coating layer are provided within the sampling opening. Thus, the outer biodegradable coating layer may dissolve when the capsule reaches a position prior to the target position (referred herein to as the priming position), and the inner biodegradable coating layer may dissolve when the capsule reaches the target position. Dissolution of the inner biodegradable coating may allow enteric fluid to enter the capsule cavity through the opening. In some embodiments, the biodegradable coating may be disposed only within the sampling opening, thereby confining the biodegradable coating within the sampling opening (not extending to the outer surface of the capsule). As will be understood by those skilled in the art, the capsule according to this disclosure may include any preferred biodegradable coating. For example, suitable coating materials include, but are not limited to, pH-sensitive polymer materials such as basic butylated methacrylate (EUDRAGIT EPO), polymethacrylate-coethyl acrylate (EUDRAGIT L 100-55), polymethacrylate-comethyl methacrylate (EUDRAGIT L100), hydroxypropyl methylcellulose phthalate (HP-55), hypromellose phthalate (HPMCP), cellulose acetate phthalate (CAP), and polyvinyl acetate phthalate (PVAP).

[0025] In some embodiments, the biodegradable coating is formed using a solution casting technique. In such a technique, a polymer is dissolved in a solution, and the solution is coated onto the cap. In some embodiments, the cap of the sampling capsule is placed on a silicone (e.g., PDMS) holder, which blocks the opening of the cap while the biodegradable coating deposits on the cap, preventing the biodegradable coating from passing through the opening. The solution may have high viscosity and may dry quickly. After the coating has dried, the cap may be lifted from the holder. The solution may easily separate from the silicone holder, thus allowing the coating to remain with the cap and cover the opening of the cap. In some embodiments, the coating may be formed by drop casting, as described below, which involves dropping large droplets with controlled size and momentum, so that the large droplets spread across the surface upon impact and wet the surface. In some embodiments, a doctor blade can be used to create a film of uniform thickness. Doctor blading, also known as knife coating or blade coating, involves moving a blade over a surface (or moving the surface beneath the blade). The small gap between the blade and the substrate determines how much solution can pass through as the blade moves, ensuring that the solution is evenly distributed across the entire substrate.

[0026] In some embodiments, each layer of the biodegradable coating is formed using a drop-casting technique. In such a technique, the biodegradable coating polymer is dissolved in a solution, and the resulting solution is deposited within the sampling opening of the capsule. In some embodiments, the cap of the sampling capsule is placed on a silicone (e.g., PDMS) holder, which blocks the opening while the first solution containing the first biodegradable coating polymer is deposited within the opening, preventing the first solution from passing through the opening. Droplets of the first solution having a known size can be deposited within the opening with controlled momentum to form a first coating layer having a first thickness. In some embodiments, a second solution containing a second biodegradable coating polymer is deposited within the opening. Droplets of the second solution having a known size can be deposited within the opening with controlled momentum to form a second coating layer having a second thickness.

[0027] In some embodiments, a doctor blade can be used to create a film of uniform thickness. Doctor blading, also known as knife coating or blade coating, involves moving a blade over a surface (or over the surface beneath the blade). A small gap between the blade and the substrate determines how much solution can pass through as the blade passes, spreading the solution evenly across the substrate.

[0028] The thicknesses of the first and second layers may be the same or different. The combined thickness of the overall coating may be selected to be sufficient to prevent premature failure of the coating. Selective variation in the thickness of each layer may enable targeted sampling at specific points along the GI tube. For example, a thicker layer of the second coating may result in the second coating requiring more time to dissolve (i.e., a longer dissolution time). The layers may be designed to target a colonic segment for sampling and may be designed by selecting the thickness of the second coating to result in a dissolution time corresponding to the transit time through the target segment.

[0029] After the coating has dried, the cap can be lifted from the holder. The coating can be easily separated from the silicone holder, thus allowing the coating to remain undamaged within the cap's opening. In some embodiments, NIR can be used to accelerate and improve the drying process. During the NIR process, wavelengths can be selected for emissions that have higher absorbance in the solvent than in the polymer to facilitate solvent removal and drying of the solution. In some embodiments, NIR can be used multiple times during the drop casting process. For example, NIR may be used once to dry the first solution before depositing the second solution, and a second time to dry the second solution.

[0030] As described above, the capsule may include a sealing member that, after the sampling hydrogel absorbs the intestinal fluid sample and expands within the capsule cavity, can be pressed by the sampling hydrogel and engaged with the sampling opening of the capsule. According to some embodiments, the sealing member may be configured to provide desired gas permeability between the capsule cavity and the external environment. For example, in some embodiments, the sealing member may be formed as a polydimethylsiloxane (PDMS) membrane, which can provide gas permeability that allows natural gas exchange between the digestive tract and the inside of the capsule (i.e., the cavity), which can help maintain the natural metabolism of the sampled bacteria and promote their survival after the capsule is sealed. Other materials suitable for the sealing member include, but are not limited to, polyvinyl chloride (PVC), thermoplastic polyurethane (TPU), silyl olefin copolymer (COC), and perfluoropolyether (PFPE).

[0031] Depending on the specific embodiment, the capsules described herein may be capable of keeping viable bacteria viable for an extended period after the capsule has been sealed via a sealing member for subsequent analysis. For example, in some embodiments, the sampling hydrogel and / or sealing member may be constructed and arranged to keep viable bacteria in the sample fluid viable for at least 1 hour, at least 5 hours, at least 10 hours, at least 20 hours, or at least up to 24 hours or more (i.e., to keep the viable bacteria alive) before the sampling fluid is collected for analysis.

[0032] In some embodiments, the device may be used to collect proteins or other biomarkers to investigate the state of the body. For example, in some embodiments, the device may be used to collect calprotectin in the gastrointestinal tract to diagnose inflammatory bowel disease (IBD). Calprotectin is a protein released by neutrophils when there is inflammation in the gastrointestinal tract.

[0033] After passing through the digestive tract, the sealed capsule can be excreted by the patient and subsequently recovered for analysis of the intestinal microbiome sample contained therein. According to some embodiments, the capsule may be constructed and arranged to allow for easy dismantling after recovery, thereby enabling easy recovery of the sampling hydrogel containing the sample. For example, in some embodiments, the capsule housing may be formed from two or more housing parts that can be detachably fixed to each other to allow access to the capsule cavity. In one exemplary embodiment, the capsule housing may be formed from two capsule parts that adhere to each other via threaded joint surfaces, thereby allowing for easy dismantling of the capsule after recovery by excretion, so that the sampling hydrogel inside the capsule can be removed for future cultivation and analysis of the bacterial sample contained in the capsule. Other preferred joint surfaces include, but are not limited to, snap-fit ​​joint surfaces and friction or interference-fit joint surfaces.

[0034] In some embodiments, the various components of the capsule housing may be treated before assembly to provide a hydrophilic coating to the capsule housing. The inventors have recognized and appreciated that such treatment may help facilitate the flow of sample fluid through the sampling opening into the cavity where the sample fluid can be absorbed by the hydrogel material contained therein. In particular, a hydrophilic coating on the surface of the capsule housing can help provide continuous fluid draw from the intestine into the narrow sampling opening of the capsule. Additionally, a hydrophilic coating on the internal surface of the sampling opening may assist in the adhesion of a biodegradable coating to the sampling opening, in addition to further contributing to the fluid draw from the intestine. For example, in some embodiments, hydrophilic surface modification may be performed by activating the surface of the housing components using air plasma treatment followed by immersion in a polyethylene glycol (PEG) solution.

[0035] The hydrogel materials disclosed herein may consist of hydrophilic polymer networks capable of absorbing large amounts of water while maintaining their structure. These polymer networks are typically crosslinked via covalent bonds, hydrogen bonds, van der Waals interactions, and / or physical entanglements. The devices disclosed herein provide non-invasive sampling devices that can passively extract and secure samples from targeted locations along the digestive tract by utilizing both the absorption capacity and the mechanical properties of the hydrogels.

[0036] In some embodiments, the sampling hydrogel in the capsule may be synthesized from a combination of acrylic acid (AA) and acrylamide (AM) monomers. It should be understood that this disclosure is not limited to specific ratios of these monomers for forming the hydrogel material. For example, preferred ratios of these monomers may include, but are not limited to, 10%AA / 90%AM, 30%AA / 70%AM, 50%AA / 50%AM, 70%AA / 30%AM, or 90%AA / 10%AM. As will be discussed in more detail below, the hydrogel material can be formed by mixing these monomers with deionized (DI) water, as well as methylenebisacrylamide (MBA) as a crosslinking agent and ammonium persulfate (AP) as an initiator. While specific hydrogel materials are described herein, it should be understood that other hydrogel materials, such as hydrogels based on other acrylic polymers (e.g., acrylic acid, acrylamide, poly(N-isopropylacrylamide), poly(N-isopropylacrylamide), and / or N,N-diethylacrylamide) and / or combinations of non-acrylic polymers, may be preferred. In some embodiments, the hydrogel can be synthesized from a combination of acrylamide and N,N'-methylenebisacrylamide (MBA).

[0037] It should be understood that the capsule housings according to this disclosure may be made from any suitable biocompatible material. For example, in some embodiments, the capsule housing may be formed from a biocompatible polymer material such as a methacrylate polymer. Other suitable materials include, but are not limited to, commercially available biocompatible polymers such as Dental LT Clear, MED625FLX, and MED610, and / or other polymer materials treated with PEG to provide biocompatibility. Furthermore, it should be understood that this disclosure is not limited to any particular method for forming the capsule housing. For example, some embodiments described in more detail below utilize capsule housings formed by a 3D printing process. Other suitable manufacturing methods may include, but are not limited to, casting methods, molding methods (e.g., injection molding), or other methods that would be understood by those skilled in the art.

[0038] Depending on the specific embodiment, the capsule may have any of the following preferred dimensions. For example, a cylindrical capsule may have a length of about 9 mm to about 23 mm and a diameter of about 4.5 mm to about 10 mm. For example, in one embodiment, the capsule may have a diameter of about 9 mm and a length of about 15 mm, which is smaller than a standard 000-size gelatin capsule (having dimensions of 9.97 × 26.14 mm). Furthermore, the sampling opening formed in the capsule housing may have a diameter selected based on the size of the sealing member contained within the capsule. For example, in some embodiments, the diameter of the sampling opening can be selected to be at least 1 mm smaller than the diameter of the sealing member, which can help ensure proper sealing of the capsule by the sealing member. In one exemplary embodiment, the sampling opening may have a diameter of about 5 mm.

[0039] Certain non-limiting embodiments are described in further detail with reference to the drawings. Since this disclosure is not limited to the specific embodiments described herein, it should be understood that the various systems, components, features, and methods described in relation to these embodiments may be used individually and / or in any desired combination.

[0040] Figure 1 is a schematic diagram of the sampling capsule 102 at various positions 10, 20, 30, and 40 within the GI tube 100. Figures 2A, 2B, 2C, and 2D are schematic diagrams of the sampling capsule 102 in various operating states that may correspond to positions 10, 20, 30, and 40 in Figure 1, respectively. As shown in Figure 2A, the capsule 102 includes a biodegradable coating 106 (e.g., an enteric coating), which has an outer biodegradable coating layer 108 and an inner biodegradable coating layer 110. The biodegradable coating 106 is positioned within the sampling opening 112 of the capsule 102, thereby sealing the sampling opening 112. In this way, the biodegradable coating 106 protects the components within the capsule cavity 104, including the sampling hydrogel 114 and the sealing member 116, and delays sampling until the capsule reaches the target position within the GI tube. In some embodiments, the biodegradable coating 106 may be disposed only within the sampling opening and not on the outer surface of the capsule. As shown in Figure 2B, when the capsule reaches the priming position in the GI tube (which may be a point in the GI before the target position), the outer biodegradable coating layer 108 decomposes, exposing the inner biodegradable coating layer 110 to the GI fluid surrounding the capsule. As shown in Figure 2C, when the capsule 102 reaches the target position, the inner biodegradable coating layer 110 decomposes, allowing the GI fluid containing the sample material 118 (e.g., the bacteria, protein, chemical, and / or other potential compounds of interest) to enter the capsule, where the fluid is absorbed by the sampling hydrogel 114. Upon absorbing the fluid, the sampling hydrogel expands and inflates within the cavity 104, filling substantially the entire volume of the cavity. Additionally, as shown in Figure 2D, the expansion of the sampling hydrogel presses the sealing member 116 into contact with the sampling opening, thereby sealing the sampling opening via the mechanical force applied by the swollen hydrogel. As shown, the fluid absorbed by the sampling hydrogel may contain the sample material 118 from inside the GI tube.After the capsule is excreted, it can be dismantled to allow for the recovery of the sampling hydrogel and the bacterial sample contained therein, so that the bacteria can be used for future cultivation and analysis.

[0041] When a patient swallows an exemplary embodiment of a sampling device having two layers of biodegradable polymer coating, the device may be exposed to saliva at a pH of 7 for typically less than one minute. As the device moves toward the stomach, the pH decreases to approximately 3. The device may still be inactive because the outer enteric-coated polymer is still deionized in the acidic pH environment of the stomach. As the device moves further down the digestive tract, the pH increases, and when the device reaches the small intestine, the average pH is 6.8, and typically up to 7.5. When the pH exceeds the dissolution threshold of the outer biodegradable coating layer, the outer polymer begins to ionize and begin to dissolve. When the outer biodegradable coating layer dissolves, the inner biodegradable coating layer is exposed to the fluids of the small intestine. At this point, the device may still be inactive because the inner biodegradable coating layer is still deionized in the basic pH environment of the small intestine. When the device reaches the ascending colon and the pH suddenly drops again in the ascending colon, the inner biodegradable coating layer may begin ionizing and dissolving in the acidic environment of the colon. As the inner biodegradable coating layer dissolves, intestinal fluid may enter the cavity 104, and the hydrogel may begin swelling by absorbing the intestinal fluid, pushing the sealing member within the cavity toward the sampling opening. With the device opening sealed by the sealing member, the device can move through the rest of the GI tube without fluid exchange.

[0042] In some cases, it may take approximately 24–72 hours for the device to travel through the colon. Depending on the intended use of the device, the dissolution of the biodegradable coating and / or the elongation of the hydrogel may be adjusted to a desired target location within the small intestine. The dissolution of the biodegradable coating may be adjusted by selecting an appropriate thickness for one or more of the coating layers described above. For example, in some embodiments, the biodegradable coating may be configured to target the ascending colon, and the coating thickness for the inner biodegradable coating layer may be selected to result in complete dissolution within 2 hours after the device enters the colon. The hydrogel may then be configured to elongate within 1 hour, and the device may finally be configured to be sealed by a sealing member within 0.5 hours. In an example targeting the transverse colon, the biodegradable coating may be configured to target the ascending colon, and the coating thickness for the inner biodegradable coating layer may be selected to result in complete dissolution within 11 hours after the device enters the colon. The hydrogel may then be configured to stretch within 1 hour, and the device may finally be configured to be sealed by a sealing member within 0.5 hours. These target time intervals may be selected to allow a safety margin of the desired time length to ensure that the device is sealed before excretion. After excretion, the capsule may be recovered and dismantled, and the sample may be analyzed for further investigation.

[0043] Figure 3 is a schematic exploded view of one embodiment of a capsule 202 for passive sampling of the gastrointestinal tract. The capsule 202 includes a capsule housing 220, which includes a first housing portion 222 (also referred to herein as a cap) and a second housing portion 224. The first housing portion includes a sampling opening 212 for providing fluid communication between the outside of the capsule and a cavity 204 formed within the capsule housing 220. A biodegradable coating 206, including an outer biodegradable coating layer 208 and an inner biodegradable coating layer 210, is provided within the sampling opening 212 to seal the sampling opening 212 until the capsule 202 reaches a desired position in the GI tube. In some embodiments, the biodegradable coating may be provided only within the sampling opening, thereby confining the biodegradable coating 206 within the sampling opening (not extending to the outer surface of the capsule). The sampling hydrogel 214 and the sealing member 216 are located within the cavity 204, and as shown, the sealing member 216 is located between the sampling hydrogel 214 and the sampling opening 212. As described above, in some embodiments, the sealing member 216 may be a flexible membrane, such as a PDMS membrane, which may provide gas permeability between the cavity 204 and the outside of the capsule 202 after the sealing member has sealed the cavity. Additionally, the first and second housing portions 222 and 224 include mounting joint surfaces 226 to allow the capsule housing to be easily disassembled and the hydrogel material recovered after the sample has been collected. In the embodiment shown, the mounting joint surface 226 has corresponding threaded features formed on the first and second housing portions, and the threaded features cooperate to form a threaded joint surface. However, as described above, other mounting arrangements such as snap-fit ​​or interference fit may be preferred in other embodiments.

[0044] In the embodiments described, the sampling opening has a circular shape. However, it should be understood that the sampling opening 212 may have any preferred shape. The shape of the sampling opening 212 can determine the shape of the biodegradable coating 206 disposed within the sampling opening 212. For example, in some embodiments, the sampling opening may be square, rectangular, elliptical, or any other preferred shape.

[0045] Figures 4A to 4F show schematic diagrams of one embodiment of a method for manufacturing a capsule having multiple layers of biodegradable coating disposed within the sampling opening of the capsule. In Figure 4A, the first housing portion 322 of the capsule is placed on a fixture 330 in the direction of the indicated arrow. The fixture 330 has a sealing plate 332, which is configured to provide a liquid-tight seal with the first housing portion 322 of the capsule around the sampling opening 318 of the capsule. The sealing plate 332 may be a silicone material (e.g., PDMS) or any other material that provides a liquid-tight seal without bonding or adhering to the biodegradable coating. A liquid solution of the first biodegradable coating 308 is introduced into the opening 318. As shown in Figure 4C, the liquid-tight seal between the sealing plate 332 and the first housing portion 322 of the capsule can hold the first biodegradable coating 308 within the opening. The first biodegradable coating 308 is then dried. In some embodiments, drying may involve the use of NIR, as indicated by arrow 334 in Figure 4C. As will be discussed below, any suitable parameters for the use of NIR may be appropriately selected, including wavelength, operating power, and scanning speed.

[0046] In some embodiments, the wavelengths used in the NIR drying process may be 300 mm, 400 nm, 500 mm or more, and / or any other suitable wavelength. In some embodiments, the wavelengths may be 2.0 μm, 2.25 μm, 2.5 μm or less, and / or any other suitable wavelength. Combinations of the above are intended, including, for example, 300 mm or more and 2.5 μm or less, 400 mm or more and 2.25 μm or less, and / or any other suitable combination of the above. Naturally, while specific ranges for NIR wavelengths are provided above, it should be understood that other ranges, both above and below the above, are also intended, as this disclosure is not limited to this form.

[0047] In some embodiments, the operating power used in the NIR drying process may be 1 kW, 3 kW, 5 kW or more, and / or any other suitable power level. In some embodiments, the operating power may be 8 kW, 10 kW, 15 kW or less, and / or any other suitable power level. Combinations of the above are contemplated, including, for example, 1 kW or more and 15 kW or less, 3 kW or more and 8 kW or less, and / or any other suitable combination of the above. Naturally, while specific ranges for NIR operating power are provided above, it should be understood that other ranges, both above and below the above, are also contemplated, as this disclosure is not limited to this form.

[0048] In some embodiments, the scanning speed used in the NIR drying process may be 1 m / min, 3.6 m / min, 5 m / min or more, and / or any other suitable speed. In some embodiments, the scanning speed may be 7.5 m / min, 10 m / min, 12.5 m / min or less, and / or any other suitable speed. Combinations of the above are intended, including, for example, 1 m / min or more and 12.5 m / min or less, 3.6 m / min or more and 7.5 m / min or less, and / or any other suitable combination of the above. Naturally, while specific ranges for NIR scanning speeds are provided above, it should be understood that other ranges, both above and below the above, are also intended, as this disclosure is not limited to this form.

[0049] As shown in Figure 4D, when the first biodegradable coating 308 has dried, the second biodegradable coating 310 can be introduced into the opening 318. The second biodegradable coating 310 is then dried. In some embodiments, drying may involve the use of NIR, as indicated by arrow 334 in Figure 4E. The first housing portion 322 is removed from the fixture, and the sealing plate 332 is separated from the first biodegradable coating 308 without damaging the coating. In some embodiments, the above method is arranged only within the sampling opening, thereby creating a biodegradable coating that is confined within the sampling opening (not extending to the outer surface of the capsule).

[0050] Figure 5 shows a schematic diagram of an alternative embodiment of capsule 402. Capsule 402 includes a biodegradable coating 406 (e.g., an enteric coating), which has an outer biodegradable coating layer 408 and an inner biodegradable coating layer 410. The biodegradable coating 406 is disposed on the outer surface 418 of capsule 402 and across the sampling opening 412 of capsule 402, thereby sealing the sampling opening 412. In this way, the biodegradable coating 406 protects the components within the capsule cavity 404, including the sampling hydrogel 414 and the sealing member 416, and delays sampling until the capsule reaches the target position in the GI tube. When the capsule reaches the priming position in the GI tube (a position that may be a point in the GI before the target position), the outer biodegradable coating layer 408 decomposes, exposing the inner biodegradable coating layer 410 to the GI fluid surrounding the capsule. When capsule 402 reaches the target position, the inner biodegradable coating layer 410 decomposes, allowing the GI fluid containing bacteria to enter the capsule, where the fluid is absorbed by the sampling hydrogel 414. Upon absorbing the fluid, the sampling hydrogel swells and expands within the cavity 404, substantially filling the entire volume of the cavity. Additionally, the expansion of the sampling hydrogel presses the sealing member 416 into contact with the sampling opening, thereby sealing the sampling opening via the mechanical force applied by the swollen hydrogel. The fluid absorbed by the sampling hydrogel may contain bacteria from within the GI tube. After the capsule is expelled, it may be dismantled to allow for the recovery of the sampling hydrogel and the bacterial sample contained therein, so that the bacteria can be used for future cultivation and analysis.

[0051] In the embodiments depicted, the biodegradable coating 406 initially covers or surrounds only a portion of the capsule 402. However, it should be understood that other arrangements, such as one in which the biodegradable coating 406 covers or surrounds the entire exterior of the capsule 402, may be preferred. In some embodiments, the biodegradable coating may have a curved outer surface. In some embodiments, the biodegradable coating may have a flat outer surface. The biodegradable coating may be in any shape that seals the sampling opening of the capsule. The biodegradable coating in Figure 5 may be deposited on the capsule by any suitable method, including drop casting, doctor blading, solution casting, or spray coating. It should be noted that any of these methods may be applied only within the sampling opening, thereby producing a biodegradable coating that is confined within the sampling opening (and does not extend to the outer surface of the capsule). [Examples]

[0052] Device Manufacturing In one embodiment, a device for passive sampling of the colon was fabricated using 3D printing. The device consisted of four components: a biodegradable coating including first and second biodegradable coating layers; a 3D printed housing including a first housing portion and a cap; a sampling hydrogel; and a gas-permeable PDMS membrane. The 3D printed housing was designed in SolidWorks (Dassault Systemes), printed using a Form 3 3D printer with stereolithography techniques, then rinsed with pure isopropyl alcohol (IPA) and UV-cured at 60°C for 15 minutes. The PDMS membrane was fabricated using a standard 1:10 ratio curing agent to silicone base and cured at 70°C for 4 hours. A circle with a diameter of 6 mm was cut in the PDMS using a computer-controlled CO2 laser cutting and engraving system (PLS6MW from Universal Laser, Inc., operating at a wavelength of 10.6 μm). A hydrogel was synthesized by dissolving acrylamide and an MBA crosslinking agent in water. The solution was then degassed by passing nitrogen gas through it for 10 minutes, and ammonium persulfate (APS) was added to the solution to act as an initiator. Finally, the solution was poured into a mold and polymerized overnight at 70°C. To prevent the device from floating in the fluid, a water-sprayed cylindrical copper disk was placed at the bottom of the housing. The copper disk was coated with PDMS to maintain the biocompatibility of the device.

[0053] Characterization of biodegradable coating layers pH-sensitive polymers include a broad category of polyacids and polybases. Polyacids can withdraw protons at low pH and release protons at either neutral or higher pH. Therefore, polyacids, such as Eudragit L100, Eudragit L100-55, and Eudragit S100, can dissolve in aqueous media at high pH but are insoluble in the lower pH range. Polybases can withdraw protons at higher pH and release protons at either neutral or lower pH. Therefore, polybases, such as chitosan and Eudragit EPO, can dissolve in aqueous media at low pH but are insoluble in the higher pH range. The release and withdrawal of protons in these polyvalent electrolytes, and the resulting solubility or insolubility, are controlled by the pH of the surrounding environment.

[0054] To better evaluate the solubility of various pH-sensitive polymers, methylene blue and a dark orange food coloring were added to solutions of Eudragit L100-55 and Eudragit EPO, respectively. These dyes enabled peak absorbance in the polymers during ultraviolet-visible (UV-Vis) spectrophotometry. The solutions were then cast onto acrylic sheets using doctor blades to obtain a uniform film thickness. After casting each layer, the film was dried in NIR (Adphos NIR-126-250 Modul, 3.6 m.) to avoid any solvent capture into the film composition. -1 The material was completely dried using a conveyor line speed and 3kW lamp power. Once the layer was dry, identical discs with a diameter of 10mm were cut using a computer-controlled CO2 laser cutting and engraving system at a wavelength of 10.6μm.

[0055] Dissolution tests of pH-sensitive polymer samples were performed at 37°C in 900 mL of various dissolving media using a USP II instrument (PTWS instrument, Pharma Test, Hainburg, Germany) at a paddle speed of 100 rpm. The dissolving media had pH values ​​of 1.2, 3, 5.5, 6.8, and 7.4. The polymer samples were removed from the dissolving media at predetermined time points, analyzed by UV-Vis using a BMG Clariostar microplate reader (BMG Labtech, Germany), and then returned to the dissolving media. The percentage of dye released from the formulation was measured using the UV-Vis technique at wavelengths corresponding to the peak wavelengths of the appropriate dyes. All tests were performed in triplicates.

[0056] The solubility tests of different polyacids (Figures 6A-6D) and polybases (Figures 7A-7C) under GI tube pH conditions showed a wide variety of solubility profiles, suggesting that diverse solubility dynamics and profiles can be achieved by different formulations.

[0057] As shown in Figures 6A-6C, all three polyacid polymers showed minimal dissolution (less than 10%) during the simulated gastric residence time (2 hours) in a simulated fasting state (pH=1.2). Continuing the experiment up to an 8-hour residence time did not result in any substantial change in this trend. Furthermore, as shown in Figures 6A-6C, all three polyacids showed less than 15% dissolution within a 2-hour gastric residence time in a simulated fasting state (pH=3.0). However, Eudragit L100-55 showed a slow, continuous dissolution trend, reaching approximately 30% after an 8-hour residence time (Figure 6A).

[0058] At a pH of 5.5, which generally simulates the pH of both the duodenum and cecum, Eudragit L100-55 showed approximately 30% dissolution after 2 hours (Figure 6A). However, Eudragit L100 and Eudragit S100 remained largely insoluble after 8 hours, showing 21% (Figure 6B) and 11% (Figure 6C), respectively. The threshold pH level for the dissolution of Eudragit L100-55 is met at a pH of 6.8, which generally simulates the proximal small intestine region. This could lead to Eudragit L100-55 being completely ionized and dissolved within 2 hours (Figure 6A). Complete dissolution of Eudragit L100 occurred after 5 hours (Figure 6B), while Eudragit S100 was only 20% dissolved after 8 hours (Figure 6C).

[0059] The solubility profiles for the polybase polymers Eudragit EPO and chitosan are shown in Figures 7A-7C. Chitosan showed relatively rapid solubility at all pH levels, with complete solubility occurring within 5-6 hours at each pH level. Eudragit EPO showed complete solubility within 1 hour at pH levels of 1.2, 3.0, and 5.5, and within 3 hours at pH 6.8. However, at pH 7.4, the solubility of Eudragit EPO did not exceed approximately 30% over the entire 8-hour residence time.

[0060] Near infrared drying In several studies, NIR technology was applied to increase the evaporation rate of the solvent and prevent mixing of polymer layers while maintaining the integrity of the polymer film structure.

[0061] Characterization of reduced cytotoxicity from NIR The in vitro cytotoxicity of samples with a bilayer biodegradable coating was determined using human mesenchymal stem cells (hMSCs) by the MTT (tetrazolium bromide) assay. Cytotoxicity was investigated both before and after NIR treatment. The cytotoxicity of capsules without a biodegradable coating was selected as a control to ensure that the partially cured 3D printed resin was non-toxic to cells. Since the coating consisted of two drop-cast layers, an investigation into the cytotoxicity of the biodegradable coating was performed. The investigation was conducted to verify whether the drop-cast layers released organic solvents as potential carcinogens and neurotoxics.

[0062] Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 100 units / mL penicillin, and 100 units / mL streptomycin. The cells were grown in a humidified incubator at 37°C, 5% CO2, and 95% relative humidity. When the cells reached confluence (approximately 80%), the samples were transferred to 24-well plates, UV-sterilized for 2 hours (1 hour per side), and then the cells were separated into 2 × 10⁶ wells per well. 4 Cells were seeded at a density of 1 cell and a final volume of 2 mL. After 24 hours of incubation, the culture medium in each well was replaced with 1800 μL of fresh DMEM and 200 μL of MTT solution (5 mg / mL thiazolyl blue in PBS), and incubated for another hour. The optical density (OD) of the mixture was measured at 490 nm using a BMG microplate reader. Cell viability was calculated using the following equation. Cell viability percentage = Number of surviving cells on the test surface / Number of surviving cells on the control surface × 100%

[0063] As shown in Figure 8, the control cell viability was calculated as 92%, indicating that the capsule housing was biologically safe. However, the cell viability of the drop-casted film dried in the environment showed a decrease to less than 50% before NIR was applied. This toxicity of the bilayer film may be mainly due to the upper layer, which, as a result of the higher molecular weight of the upper layer compared to the lower layer with a lower molecular weight, could trap a larger proportion of the solvent within the structure of Eudragit L100-55, which will be discussed in more detail below.

[0064] Cell viability increased to 84% after NIR heat treatment, suggesting that polymer dissolution was harmless to cells. The biocompatibility of the NIR-treated biodegradable coating was attributed to the evaporation of the captured solvent, which is consistent with the results of thermogravimetric analysis (TGA) discussed below.

[0065] ATR-FTIR characterization of the NIR effect To ensure that the double-layer drop casting film does not exhibit mixing effects after NIR treatment, the infrared spectra of single layers of L100-55 and EPO polymer were measured using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy with a PerkinElmer Spectrum 100 FTIR spectrometer at 4000 cm⁻¹. -1 ~550cm -1 The infrared spectra of the bilayer film were compared by scanning 50 times over a range. Single layers of Eudragit L100-55 and Eudragit EPO were evaluated in both film and powder states. Figure 9A shows the comparative ATR-FTIR spectra of pure EPO and L100-55 powder, and Figure 9B shows the comparative ATR-FTIR spectra of pure EPO and L100-55 film, as well as the bilayer coated film.

[0066] As shown in Figure 9A, the ATR-FTIR spectra for pure L100-55 and EPO powders are attributed to the C=O stretching vibration pattern of the ester group and the CO bond of the carboxylic acid ester, respectively, at approximately 1700 cm⁻¹. -1 and 1100cm -1 The main characteristic peaks were demonstrated at [location]. Additionally, the spectrum for Eudragit EPO is approximately 2850 cm⁻¹. -1 The stretch band was shown. This band may be for the dimethylamine group that distinguishes L100-55 from EPO.

[0067] As shown in Figure 9B, the ATR-FTIR spectrum of the film showed significant peaks for both L100-55 and EPO without any significant additional vibrational bands for solvent residue. These observations demonstrate that NIR treatment is an effective drying technique that can remove all solvent without affecting the polymer film structure.

[0068] Furthermore, as shown in Figure 9B, the ATR-FTIR spectrum for the bilayer coating film demonstrated characteristic peaks for the pure L100-55 polymer, without any prominent peaks indicating the mixing of the EPO layer into the L100-55 film. Since the top coating was a pure L100-55 film, this observation confirms that the expected separation of the two polymer films could have occurred due to the miscible behavior of the initial polymer solution.

[0069] TGA Characterization of NIR Effects Thermal characterization of Eudragit L100-55 (shown in Figure 10A) and Eudragit EPO (Figure 10B) in both powder and film forms was performed using thermogravimetric analysis (TGA). In one film form, the film was dried in the environment. In the other film form, the film was dried using NIR. No solvent was used in the powder form.

[0070] The difference in weight loss between the powder and film configurations may be due to the loss of moisture and solvent present in the film configuration, as these factors are absent in the powder configuration. Furthermore, films dried in the environment may capture a higher percentage of solvent than films dried using NIR. As shown in both Figures 10A and 10B, NIR drying can remove a larger percentage of captured solvent than environmental drying, resulting in a TGA curve that is closer to the TGA curve of the powder configuration.

[0071] In all cases of Eudragit L100-55 (Figure 10A), weight loss at 50–150°C may be due to evaporation of excess solvent, and loss at 225–425°C may be due to thermal decomposition of the polymer structure. For Eudragit EPO in Figure 10B, loss at 275–325°C may be a result of solvent evaporation, and the larger decrease at 375–450°C may be due to thermal decomposition.

[0072] NIR demonstrates a significantly greater effect on Eudragit L100-55 than on Eudragit EPO. This may be due to the higher molecular weight of Eudragit L100-55. With a molecular weight of 320,000 g / mol, Eudragit L100-55 is considerably heavier and more viscous than Eudragit EPO, which has a molecular weight of only 47,000 g / mol. This higher molecular weight can cause substantially more entanglement and capture of solvent particles, potentially making it more difficult to remove the solvent particles without the aid of drying techniques, such as NIR. As a result, the difference between Eudragit L100-55 dried by NIR and Eudragit L100-55 dried in the ambient environment was observed to be significantly greater than the difference between Eudragit EPO dried by NIR and Eudragit EPO dried in the ambient environment. Since radiation from NIR is not destructive to the polymer structure, solvent molecules can be targeted for evaporation without affecting the film structure or the capsule body.

[0073] Microscopic characterization of NIR effects To visualize the effect of NIR on the morphology and miscibility of bilayer biodegradable coatings, cross-sectional images of the bilayer coatings were taken. Since both solvents, Eudragit L100-55 and Eudragit EPO, were substantially identical, a high degree of polymer miscibility was possible when the layers were slowly dried under environmental conditions. In some cases, the mixed polymers may result in unintended dissolution profiles at various pH levels. For example, Figure 11A shows bilayer formation when the upper and lower layers are dried in the ambient environment. The mixture shown in Figure 11A confirms that the two polymers are blended with each other. In this configuration, the bilayer coating may not exhibit the intended response to various pH levels. The blending of the layers may have occurred partly because the slow drying process of the upper layer allowed the solvent to dissolve the lower layer. This effect, where the application of the upper layer can cause the dissolution of the lower layer over time, can lead to polymer miscibility.

[0074] Figure 11B shows the composition of Figure 11A after 2 hours of exposure to a pH level of 1.2. Under these conditions, the upper layer may not have been able to protect the lower layer from dissolution as a result of mixing of the two layers. Thus, the orange layer completely dissolved, and the thickness of the blue layer decreased significantly. Figure 11C shows that the remaining upper layer completely dissolved after 6 hours at a pH of 6.8. This dissolution of the upper layer should be expected due to a pH level exceeding the pH dissolution threshold of the upper layer polymer (Eudragit L100-55). Figure 11D simply shows that there was no lower layer left to dissolve at a pH level of 5.5 because the lower layer had already dissolved as a result of mixing.

[0075] Figures 11E–11H show the effect of NIR treatment on a bilayer coating. NIR treatment was used to prevent polymer blending through rapid and effective drying of the film. As shown in Figure 11E, the lower and upper layers were more clearly separated and better laminated as individual layers. The layers showed virtually no dissolution or change in thickness after immersion in pH 1.2 for 2 hours (Figure 11F). This may be because the pH did not exceed the dissolution threshold of the upper layer according to Eudragit L100-55. Thus, the upper layer could be preserved, acting as a shield to prevent the lower layer from being exposed to a lower pH environment that would cause the lower layer to dissolve. However, as shown in Figure 11G, the dissolution threshold of the upper layer is met when the pH rises to 6.8. Figure 11G shows the complete dissolution of the upper layer after 6 hours at a pH level of 6.8. When the upper layer dissolved, the lower layer was exposed to a higher pH environment. As shown in Figure 11H, the lower layer was then completely ionized and dissolved after the pH was reduced to 5.5 for 1 hour.

[0076] SEM Characterization of NIR Effects Additionally, high-magnification cross-sectional images of both the bilayer biodegradable coatings before and after NIR treatment are shown in Figures 12A and 12B. These images were captured using a scanning electron microscope (SEM) (Hitachi-S 4800, Tokyo, Japan). As shown in Figure 12A, the upper layer (Eudragit L100-55 dried in ambient conditions) showed significant porosity. The lower layer (Eudragit EPO dried in ambient conditions) showed significantly less porosity than L100-55. These pores may be formed as a result of a slow drying process under ambient conditions. Additionally, as mentioned above, the higher molecular weight of Eudragit L100-55 can cause a greater degree of entanglement between the polymer chains and particles of both solvents and the air, effectively preventing solvent and air particles from easily escaping the polymer structure.

[0077] Figure 12B shows that when the coating layer is dried using NIR, there is no similar porosity problem in the Eudragit L100-55 upper layer. This may be a result of the pulsed heat generated by the lamp energizing solvent and air particles, thereby forcing bubbles to escape from the polymer matrix, ultimately resulting in a polymer structure free of pores and cracks. In short, microscopic and SEM cross-sectional images after NIR treatment can demonstrate the efficiency of this technique, not only achieving proper dissolution and protection in different pH environments but also supporting acceptable polymer morphology.

[0078] Bacterial sampling In vitro research To evaluate the sampling capabilities of both the hydrogel and the overall device, four containers were prepared to hold mixtures of gut microbiota. Each container contained a different mixture, as shown in Table 1 below. The composition of the samples drawn in by each device and each hydrogel was compared to the composition of the initial mixture. The bacteria used included Lactobacillus cremoris (Firmicutes), Bacteroides fragilis (Bacteroidetes), Escherichia coli LF82 (Proteobacteria), and Akkermansia muciniphila (Verrucomicrobia), as shown in Table 2. The bacterial strains were cultured overnight at 37°C in an anaerobic chamber with their respective broths, also shown in Table 2.

[0079] [Table 1]

[0080] [Table 2]

[0081] Separately, the fully assembled device and the free-swelling hydrogel were immersed in a container for 2 hours while incubating at 37°C at 100 rpm. Next, the complete device was disassembled. The disassembled components and the free-swelling hydrogel were transferred to a separate vial containing 4 mL of DI water and extracted in a stirrer at 100 rpm for 2 hours.

[0082] After a 2-hour extraction, DNA extraction was performed in extraction buffer (DI water) using the Invitrogen® PureLink® Genomic DNA Mini-Kit (Invitrogen, USA). Each bacterial mixture was aliquoted before ("pre") and after ("post") sampling with the device or hydrogel. Table 1 shows that all bacterial compositions sampled by the complete device were similar to the corresponding mixtures (both pre and post). While the results for hydrogel alone were less representative, this in vitro bacterial sampling experiment confirmed the ability of the capsule device to capture representative samples of the gut microbiota.

[0083] In vivo research In vivo evaluation of device functionality was performed using pigs. The experiment was approved by the Purdue University Animal Care and Use Committee according to protocol number 1911001975A002. Prior to the experiment, the pigs were weighed (each approximately 40-50 kg) and individually housed in metabolic crates. The pigs were subjected to a 5-day acclimatization period in their new environment and given free access to water. Their diets, based on corn and soy meal, were formulated to meet or exceed NRC (2012) requirements. The pigs were fed a daily level of three times their estimated energy maintenance requirement (i.e., 197 kcal ME / BW 0.60 kg; approximately 4% of pig body weight per day), and their daily feed allocation was provided every morning.

[0084] The pigs were fasted for 12 hours before capsule administration. Two hours after capsule administration, the pigs were fed a diet containing a refractory ferric oxide marker, and the rate of digestion was determined. The pigs were then observed at 30-minute intervals to determine the length of time required for the marker to be excreted in the feces (red).

[0085] The capsules were administered orally using a dispensing gun designed for use in pigs. Each pig was euthanized upon excretion and collection of the capsules, and the pH of various sections within the GI tube was measured. To avoid fluid homogenization and potential errors in pH values, the stomach, small intestine, and large intestine were isolated using cable ties. To achieve more accurate profiling, the small intestine was segmented into 2-meter increments (Duo: duodenum, J: jejunum, Ile: ileum, used in Figures 13A and 13B), and the large intestine was segmented into 1-meter increments (LI: large intestine, used in Figures 13A and 13B). The fluid contents of each section were transferred to disposable plastic weighing boats, and pH values ​​were obtained from these fluid contents.

[0086] The administered capsules were recovered from the animals after excretion. After wiping away any external fecal matter and rinsing, the capsules were disassembled. The disassembled components were transferred to a separate vial containing 4 mL of sterile DI water and extracted in a stirrer at 100 rpm for 2 hours.

[0087] For each lumen content sample, DNA was extracted using the QIAamp PowerFecal Pro DNA Kit (Qiagen, Germany) according to the manufacturer's recommendations. The DNA concentration and quality of each sample were measured using a NanoPhotometer NP60 (Implen, Germany).

[0088] For all DNA extraction samples, the V4 region of the 16S rRNA gene was amplified using PCR primers 515 forward, 5'GTGCCAGCMGCCGCGGTAA, and 806 reverse, 5'GGACTACHVGGGTWTCTAAT. PCR reactions were performed individually in 50 μL total volume using Thermo Scientific® Phusion High-Fidelity PCR Master Mix (Thermo Scientific, USA) as recommended by the manufacturer. The expected amplicon size (approximately 254 bp) was verified by 2% agarose gel electrophoresis. The PCR products were then purified using Invitrogen® PureLink® PCR Purification Kit (Invitrogen Life Technologies, USA).

[0089] After confirming sample quality using a NanoPhotometer NP60, PCR products were sent to the Purdue Genomics Core Facility for WideSeq (next-generation sequencing) analysis. Sequencing libraries were prepared using the Nextera DNA Library Preparation Kit (Illumina, CA, USA), and the multiplexed libraries were sequenced using MiSeq (Illumina, CA, USA) to generate paired-end reads (2 × 250 bp). Paired-end reads from each sample were processed to remove adapters and low-quality bases. Only selected paired reads were used for further processing. The reads were then newly reconstructed into complete sequences and taxonomically assigned using the Silva rRNA database.

[0090] The V4 region of 16S rRNA was amplified and sequenced from 17 different luminal contents samples from pigs 1 and 2. An average of 52,413 total reads were obtained per sample using a MiSeq instrument (Illumina, CA, USA). The sequences were processed to remove adapters and low-quality bases. The selected reads were newly reconstructed into complete sequences, taxonomically assigned using the Silva rRNA database, and aggregated into 12 phyla. The results were used to construct microbial taxonomic composition profiles for pigs 1 and 2.

[0091] The results shown in Figures 13A and 13B demonstrated that the microbiome composition can vary significantly along the pig intestine, particularly between the small and large intestines. The most dominant phylum throughout the intestine was Firmicutes (64%–99%). The Bacteroidetes phylum increased in the large intestine segment, representing 19%–32% of total bacteria. The difference between the Bacteroidetes / Firmicutes ratio in the small intestine and the Bacteroidetes / Firmicutes ratio in the large intestine could be used as a biomarker to indicate the device sampling location.

[0092] Furthermore, two devices administered to pig 1 and pig 2 were collected and analyzed using the same process via WideSeq. Capsule 1 from pig 1 was found in pig 1's feces. Capsule 2 from pig 2 was not excreted by the time of euthanasia and was found in the colon sample while dissecting the GI tube. Capsule 1 had Firmicutes and Bacteroidetes compositions of 73% and 24%, respectively. This result was very similar to the colon profile of pig 1 (75–78% and 19–21%, respectively). Capsule 2 had a lower Bacteroidetes composition (4%). However, the Bacteroidetes composition was still significantly higher than the Bacteroidetes composition in the small intestinal segment of pig 2 (0.02–0.06%). This indicates that the sampling device drew the sample from the colon as intended and expected.

[0093] Although these teachings have been described in conjunction with various embodiments and examples, they are not intended to be limited to such embodiments or examples. Rather, the teachings of the present invention encompass a variety of alternatives, modifications, and equivalents, as will be understood by those skilled in the art. Accordingly, the foregoing descriptions and drawings are for illustrative purposes only.

Claims

1. A method for manufacturing a device (102, 202) for passive sampling of the gastrointestinal tract, A capsule housing (220) having a first housing portion (222, 322) and a second housing portion (224) that cooperate to form a cavity, wherein the first housing portion has sampling openings (112, 212, 318), and the sampling openings provide fluid communication between the cavity and the outside of the capsule housing; and, The sampling opening is closed with a biodegradable coating (106, 206, 308) comprising multiple biodegradable coating layers, wherein the decomposition of the multiple biodegradable coating layers exposes the sampling opening, allowing fluid flow into the cavity, and the closure is performed accordingly. Closing down The first housing portion is placed on the sealing plate (332) of the jig (330), such that the sealing plate forms a liquid-tight seal with the peripheral edge of the sampling opening. The first biodegradable coating layer (108, 208, 308) and the second biodegradable coating layer (110, 210, 310) are deposited within the sampling opening using a drop casting technique, wherein the first and second biodegradable coating layers are confined within the sampling opening, and Remove the first part of the capsule housing from the sealing plate. A method characterized by including the following.

2. The drop casting technique is, Dissolving an enteric-coated polymer in a liquid solvent to form a polymer solution, and The method according to claim 1, comprising depositing droplets of a polymer solution having a controlled size onto the surface of a sealing plate within a sampling opening to form a first biodegradable coating layer.

3. The drop casting technique further includes: After forming a first biodegradable coating layer by droplet deposition, the polymer solution is dried using near-infrared light to remove the solvent, and A second biodegradable coating layer is formed by depositing droplets of a polymer solution having a controlled size onto the surface of a first biodegradable coating layer within a sampling opening. The method according to claim 2, including the method described in claim 2.

4. The method according to claim 3, further comprising drying the polymer solution of the second biodegradable coating layer using near-infrared light to remove the solvent from the second biodegradable coating layer.

5. The method according to claim 1, further comprising drying a plurality of biodegradable coating layers using near-infrared light.

6. The method according to claim 5, wherein the near-infrared light has a wavelength of 400 mm to 2.25 μm and an operating power of 3 kW to 8 kW.

7. The method according to claim 1, wherein the first and second housing portions are attachable to each other via a screw-in interface or a snap-fit ​​interface.

8. The method according to claim 1, wherein the first biodegradable coating layer is configured to decompose when exposed to a first pH level, and the second biodegradable coating layer is configured to decompose when exposed to a second pH level different from the first pH level.

9. The method according to claim 1, further comprising providing a sampling hydrogel in a cavity, wherein the sampling hydrogel is configured to absorb the sample fluid upon exposure to the sample fluid, expand in the cavity, and store the sample fluid for subsequent analysis.

10. The method according to claim 9, wherein the apparatus includes a sealing member (116) disposed in a cavity between the sampling hydrogel and the sampling opening, wherein the expansion of the sampling hydrogel in the cavity pushes the sealing member, causing the sealing member to engage with the sampling opening and seal the cavity.

11. The method according to claim 10, wherein the sealing member is made of a gas permeable membrane.

12. The method according to claim 1, comprising closing the sampling opening using a first biodegradable coating layer, drying the first biodegradable coating layer using near-infrared light, depositing a second biodegradable coating layer on top of the first biodegradable coating layer, and drying the second biodegradable coating layer using near-infrared light.

13. The method according to claim 1, wherein the sampling hydrogel is synthesized from acrylic acid and acrylamide monomer.

14. The method according to claim 13, wherein the ratio of acrylic acid monomer to acrylamide monomer is about 10% acrylic acid monomer and about 90% acrylamide monomer.

15. The method according to claim 1, wherein the sealing plate is made of silicone.

16. The method according to claim 1, further comprising applying a hydrophilic coating to the inner surface of the sampling opening.

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