Gastrointestinal sampling capsule

Abstract

A passive device for non-invasive gastrointestinal sampling is disclosed. In some embodiments, the capsule device includes a capsule housing having a sampling aperture and a biodegradable coating sealing the sampling aperture. A biodegradable coating may be disposed within the sampling aperture. The capsule comprises an absorbent sampling hydrogel and a sealing member located between the sampling hydrogel and the sampling aperture. The capsule is taken by a patient. Once the capsule reaches a desired location in the gastrointestinal tract, the biodegradable coating dissolves, causing gastrointestinal fluid in the capsule to be absorbed by the sampling hydrogel. Absorption of the fluid causes the sampling hydrogel to expand, thereby pressing the sealing member toward the sampling aperture to seal the capsule. In some embodiments, the biodegradable coating may have multiple layers that can dissolve at different locations of the gastrointestinal tract to facilitate targeting specific locations, such as the colon.

Description

[0001] field

[0002] The disclosed embodiments relate to non-invasive gastrointestinal tract sampling capsules and related methods of use.

[0003] background

[0004] Developing new tools that can accurately sample microbial communities, proteins, chemicals, and other biomarkers of interest throughout the gastrointestinal tract (GI tract) could aid in more efficient and accurate prediction and diagnosis of disease and disease progression.

[0005] Many studies have found that the gut microbiota plays an important role in the pathophysiology that dominates human health. Many studies have identified the impact of the gut microbiota on human metabolism, nutrient uptake, the efficacy of orally administered therapies, and immune and nervous system function. For example, many studies have found correlations between microbial imbalance (dysbiosis) and a variety of diseases, including diabetes, obesity, and metabolic syndrome; these diseases affect approximately 30 million people in the United States. Similarly, recent insights into possible pathways by which gut bacteria may affect the development and maintenance of the nervous system suggest a link between gut microbiota composition and the regulation of psychiatric neurological disorders, including dysbiosis in anxiety, depression, and autism.

[0006] In addition, many diseases are associated with the production of different biomarkers and mRNA transcripts in the gastrointestinal tract. For example, fecal calprotectin (fCal) has been shown to be the most accurate in detecting inflammatory bowel disease (IBD) and mucosal damage. Although some studies have demonstrated an association between fCal levels and the degree of inflammation, the levels of calprotectin are highly dependent on several factors, including diet, water content in the stool sample, and the location of the disease. Patients with ileal Crohn's disease (CD) may have large ulcers and low fCal levels. This may indicate that although stool analysis can simply evaluate potential IBD, it does not indicate possible disease location or status.

[0007] Overview

[0008] In one embodiment, a device for passive sampling of the gastrointestinal tract includes a capsule shell surrounding a cavity, a sampling hole formed in the capsule shell and providing fluid communication between the cavity and the outside of the capsule shell, and a sampling hydrogel located inside the cavity. When exposed to a sample fluid, the sampling hydrogel is configured to absorb the sample fluid, expand in the cavity, and store the sample fluid for subsequent analysis. The device also includes a biodegradable coating covering the sampling hole. The biodegradable coating includes a plurality of biodegradable coating layers. The degradation of the plurality of biodegradable coating layers exposes the sampling hole to allow fluid to flow into the cavity.

[0009] In another embodiment, a method for preparing a passive sampling device for the gastrointestinal tract includes providing a capsule shell having a capsule cavity and a sampling hole, wherein the sampling hole provides fluid communication between the capsule cavity and the outside of the capsule shell. The method also includes closing the sampling hole with a biodegradable coating comprising multiple biodegradable coating layers. Degradation of the multiple biodegradable coating layers exposes the sampling hole to allow fluid to flow into the capsule cavity.

[0010] In another embodiment, a device for passive sampling of the gastrointestinal tract includes a capsule shell defining a capsule cavity, a sampling hole formed in the capsule shell and providing fluid communication between the capsule cavity and the outside of the capsule shell, and a sampling hydrogel located inside the capsule cavity. When exposed to a sample fluid, the sampling hydrogel is configured to absorb the sample fluid, expand in the capsule cavity, and store the sample fluid for subsequent analysis. The device also includes a sealing member in the capsule cavity between the sampling hydrogel and the sampling hole. The expansion of the sampling hydrogel in the capsule cavity presses the sealing member to engage with the sampling hole to seal the capsule cavity. The device also includes a biodegradable coating located in the sampling hole. Degradation of the biodegradable coating exposes the sampling hole to allow fluid to flow into the capsule cavity.

[0011] It should be understood that the aforementioned concepts and other concepts discussed below can be arranged in any suitable combination, as the present disclosure is not limited in this regard. In addition, other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments when considered in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The drawings are not drawn to scale. In the drawings, each identical or nearly identical component shown in various figures may be represented by a like number. For clarity, not every component may be labeled in every figure. In the drawings:

[0014] Figure 1 is a schematic diagram showing the passage of a capsule through the gastrointestinal tract according to some embodiments;

[0015] Figure 2A An embodiment of a capsule positioned at a first location in the gastrointestinal tract is shown;

[0016] Figure 2B Shown is a second location in the gastrointestinal tract Figure 2A Capsules in;

[0017] Figure 2C The third location in the gastrointestinal tract is shown. Figure 2A Capsules in;

[0018] Figure 2D The fourth position in the gastrointestinal tract is shown. Figure 2A Capsules in;

[0019] Figure 3 is a partially exploded view of a capsule according to some embodiments;

[0020] Figure 4A is a schematic diagram showing the first step of a method for preparing a capsule coating using a drop casting technique, according to some embodiments;

[0021] Figure 4B Yes Display Figure 4A Schematic diagram of the second step of the method;

[0022] Figure 4C Yes Display Figure 4A Schematic diagram of the third step of the method;

[0023] Figure 4D Yes Display Figure 4A Schematic diagram of the fourth step of the method;

[0024] Figure 4E Yes Display Figure 4A Schematic diagram of the fifth step of the method;

[0025] Figure 5 An alternative embodiment of the capsule is shown;

[0026] Fig. 6A The dissolution curve of Eudragit L100-55 at different pH levels is shown;

[0027] Figure 6B The dissolution curve of Eudragit L100 at different pH levels is shown;

[0028] Figure 6C The dissolution curves of Eudragit S100 at different pH levels are shown;

[0029] Fig.6D is a bar graph showing the dissolution percentage of the biodegradable coating polymer after exposure to different pH levels for 2 hours;

[0030] Fig. 7A The dissolution curve of Eudragit EPO at different pH levels is shown;

[0031] Figure 7B The dissolution curves of chitosan at different pH levels are shown;

[0032] FIG7C is a bar graph showing the percent dissolution of a biodegradable coating polymer after exposure to different pH levels for 1 hour;

[0033] Figure 8is a bar graph showing the cytotoxicity of the biodegradable coating before and after NIR drying;

[0034] Fig.9A Shown are the spectroscopic results for the enteric polymer in powder form;

[0035] Fig. 9B The spectral results of the enteric polymer film are shown;

[0036] Fig. 10A Thermogravimetric analysis of various forms of Eudragit L100-55 is shown;

[0037] Fig. 10B Thermogravimetric analysis of various forms of Eudragit EPO is shown;

[0038] Fig.11A is a microscopic image of a double-layer biodegradable coating dried in ambient environment;

[0039] Fig. 11B yes Fig.11A Microscopic image of the double-layer biodegradable coating after exposure to pH 1.2 for 2 hours;

[0040] Fig. 11C yes Fig. 11B Microscopic image of the double-layer biodegradable coating after exposure to pH 6.8 for 6 hours;

[0041] Fig.11D yes Fig. 11C Microscopic image of the double-layer biodegradable coating after exposure to pH 5.5 for 1 hour;

[0042] Fig.11E is a microscopic image of a double-layer biodegradable coating dried using NIR;

[0043] Fig.11F yes Fig.11E Microscopic image of the double-layer biodegradable coating after exposure to pH 1.2 for 2 hours;

[0044] Fig.11G yes Fig.11F Microscopic image of the double-layer biodegradable coating after exposure to pH 6.8 for 6 hours;

[0045] Fig.11H yes Fig.11G Microscopic image of the double-layer biodegradable coating after exposure to pH 5.5 for 1 hour;

[0046] Fig. 12A is a SEM image of a double-layer biodegradable coating dried in ambient environment;

[0047] Fig. 12B is a SEM image of a double-layer biodegradable coating dried using NIR;

[0048] Fig.13A is a bar graph showing the composition of the gastrointestinal microbiota compared to the capsules administered to the first animal; and

[0049] Fig. 13B is a bar graph showing the composition of the gastrointestinal microbiota and comparison with capsules administered to a second animal.

[0050] Details

[0051] At least some aspects described herein relate to targeted sampling of microbial communities, proteins, chemicals, and other biomarkers throughout the gastrointestinal tract.

[0052] Sampling the human microbiota is becoming an important aspect of understanding the mechanisms of microbiota-drug interactions and the extent to which this complex interaction affects drug efficacy and bioavailability. Most of the known information about the structure and function of the human gut microbiota has been determined by ex situ culture and / or bacterial sequencing in fecal samples. However, only a small fraction of intestinal bacteria can be obtained and cultured from fecal samples, so efforts have been made to develop tools that can sample microorganisms directly from the gastrointestinal (GI) tract. For example, colonoscopy and / or gastroscopy methods are currently used, but these methods are limited to sampling certain parts of the entire GI tract and are invasive methods that cause patient discomfort and lead to decreased compliance. Other methods have used smart functional capsules that are able to collect samples at different target locations in the GI tract, and these methods can address some of the limitations of traditional colonoscopy and gastroscopy. In addition, capsule-based devices can improve patient comfort without the need for administration in a clinical setting. For example, the PillCam TM Capsule endoscopy (CE) technology is used to collect images of hard-to-reach areas of the gastrointestinal tract to diagnose diseases related to the small intestine, such as unexplained gastrointestinal bleeding, tumors, Crohn's disease, angiodysplasia, celiac disease, and polyposis. However, the technology lacks the ability to collect and store samples as it passes through the gastrointestinal tract.

[0053] Efforts to develop new capsules to sample the intestinal microbiota using different approaches can be divided into two major categories: active and passive devices. In active devices, the drive and sampling mechanism is usually achieved by using an on-board battery of the device, which provides the required energy to drive a variety of plungers, pistons, biopsy forceps, etc., which collect and store the sample in the capsule. However, in such devices, the battery often occupies a large portion of the capsule volume, which may limit the space for sample storage, and active devices generally present a high risk of failure and the possibility of corrosive electrolyte leakage, which can cause severe corrosive damage and liquefaction necrosis. In order to avoid these and other disadvantages associated with active devices, a variety of passive drive sampling mechanisms have been developed to make the capsule more compact, economically feasible, and less safety-related issues. In such designs, the capsule passes through the gastrointestinal tract by peristaltic motion at an average speed of 1-2 cm / min, and collects the sample by simple passive drive (such as capillary wicking or pressure difference). However, the inventors have recognized and understood that there are many important design considerations that existing passive devices cannot address. For example, some existing methods rely on capsule assembly, requiring a well-sealed vacuum chamber within the capsule, which significantly increases the complexity of the device. Other methods rely on retrieving the sampling device via a string extending upstream from the gastrointestinal tract, which may cause discomfort to the patient.

[0054] In some embodiments, a capsule is provided, the capsule having a hole, the hole forming an opening from the outside of the capsule to the inside. The hole can be covered with a pH-sensitive enteric polymer coating, which dissolves when entering the basic environment of the small intestine. In some embodiments, the inside of the capsule is a hydrogel, which draws the fluid sample into the capsule. When the fluid enters the capsule, the hydrogel swells. An elastomeric disk can be provided for the hydrogel. When the hydrogel swells, the elastomeric disk can be pushed toward the hole to seal the capsule from the inside, thereby preventing any further fluid exchange.

[0055] The present inventors have recognised that, in certain circumstances, providing a biodegradable coating on the outside of the capsule to cover the opening may require the capsule to have a corner or collar near the aperture to contain the liquid solution during application.

[0056] Therefore, the inventors have recognized that, in some embodiments, it may be beneficial to place a biodegradable coating in the capsule hole rather than outside the capsule. Placing a biodegradable coating in the hole can make the capsule outside present a wider range of geometric shapes, including smoother and more rounded geometric shapes, which may be more suitable for passing through the gastrointestinal tract. For example, the capsule can be an elongated oval, with only a small hole formed at one end. The biodegradable coating can be placed only in the hole, so that the capsule outside has only a circular feature. Alternatively or in addition, in some embodiments, placing a biodegradable coating in the hole can increase the ease of manufacturing. For example, in some embodiments, it may be easier to create a coating layer by depositing a coating material in the closed volume of the hole than applying a coating material around the outer surface of a circular capsule. In some embodiments, depositing a coating material in the closed volume of the hole can help control the coating layer thickness. This may be particularly beneficial when applying multiple coating layers, as described below.

[0057] Another limitation of current designs is that they target only the small intestine. Biodegradable coatings must resist degradation in an acidic environment in order to survive in the stomach, but must dissolve in an alkaline environment in order to allow sampling in the alkaline environment of the small intestine. Because the large intestine is more acidic, no single pH-sensitive coating has been determined to resist both the acidic environment of the stomach and the alkaline environment of the small intestine to reach the colon intact and dissolve therein. This limitation of enteric polymer coating materials has inhibited the development of similar colon passive sampling devices. As described above, currently available colon sampling techniques are typically invasive, expensive, time-consuming, and require skilled technicians. Therefore, there is still an unmet need for user-friendly targeted passive sampling from the colon.

[0058] The inventors have recognized that in some embodiments, it may be beneficial to include a multilayer biodegradable coating, wherein each layer dissolves at a different pH level. This arrangement can 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 can dissolve in an alkaline environment and the inner layer can dissolve in an acidic environment. The outer layer can survive passing through the stomach and protect the inner layer from the acidic environment therein. The outer layer can dissolve in the alkaline environment of the small intestine, thereby exposing the inner layer. The inner layer can survive in the small intestine to dissolve in the acidic environment of the colon, thereby allowing the passive sampling process to occur in the colon.

[0059] The inventors have recognized that in some embodiments, it may be useful to use a drop casting method to form one or more biodegradable coatings. The inventors have recognized that drop casting can be used to produce thick layers in a shorter period of time while maintaining control and simplicity of the application process. For example, an enteric polymer can be dissolved in a liquid solvent to form a polymer solution. Droplets of a polymer solution with a controlled or known size can be deposited on a surface or in a hole with controlled momentum, thereby quickly producing a coating layer with a known thickness. However, it should be understood that in other embodiments, other methods of forming a biodegradable coating can be used.

[0060] The inventors also recognize that, in some cases, when the solvent used to produce the polymer solution is toxic or otherwise bioincompatible, the biocompatibility of the coating may be impaired if the solvent is trapped inside the polymer matrix. In addition, the solvent may act as a plasticizer in the polymer matrix, thereby potentially reducing the rigidity of the coating layer and potentially increasing the possibility of premature rupture of the sealing member. Therefore, the inventors have recognized that, in some embodiments, it may be desirable to remove the solvent from the coating layer after deposition.

[0061] The inventor has recognized that, in some embodiments, it may be useful to use near infrared (NIR) spectroscopy to dry polymer solution and remove solvent after deposition. In the NIR process, the wave of known and controlled wavelength is emitted on the sample material. Although NIR is usually used as a method for identifying unknown materials, the ability of controlling the emission wavelength allows the selection of a wavelength with higher absorbance than in enteric polymers in the solvent. Higher absorbance in the solvent may cause solvent evaporation, and lower absorbance in the enteric polymer may cause the polymer film structure to be almost undamaged or undamaged. For example, the NIR method can allow solvent to evaporate from the polymer matrix in a relatively short time, thereby removing the solvent before the polymer matrix is ​​destroyed. However, it should be understood that in other embodiments, other methods for removing the solvent may be used, such as using heating.

[0062] Additionally, in designs with multiple layers of biodegradable coatings, the ability of each layer to dissolve at a desired point in the gastrointestinal tract may rely on the separation or stratification of the different biodegradable coatings into different layers. When the different biodegradable coatings are allowed to mix, the effect is similar to creating a single layer with a concentration gradient running through it. The result is that the entire coating may partially dissolve in each part of the gastrointestinal tract, potentially compromising the ability of the capsule to specifically target a particular point in the gastrointestinal tract (e.g., the colon). To prevent mixing, each layer of coating may be dried or cured after deposition and before any subsequent layers are deposited.

[0063] In view of the above, the inventors have recognized that in some embodiments, it may be beneficial to apply NIR to the coating layer before applying subsequent layers. For example, NIR can be used to quickly dry and remove solvent from the first polymer coating after the first polymer coating layer is drop-cast in the hole of the sampling capsule and before the second polymer coating layer is drop-cast on the first layer. The NIR can then be used to quickly dry and remove solvent from the second layer to obtain a biocompatible coating with multiple structurally intact different layers. However, it should be understood that in other embodiments, other methods for achieving curing can be used, such as using heating.

[0064] In some embodiments, the passive sampling device may include a capsule that the patient can ingest so that the capsule passes through the gastrointestinal tract. The capsule may include a capsule shell defining a capsule cavity and an adsorption sampling hydrogel located in the capsule cavity. The capsule shell may include a sampling hole that allows fluid from the gastrointestinal tract (e.g., fluid containing microorganisms or proteins such as calprotectin) to flow into the capsule cavity and be absorbed by the sampling hydrogel in the capsule cavity. When absorbing the fluid, the sampling hydrogel can expand in the capsule cavity and press a sealing member (e.g., a sealing membrane) in the capsule cavity between the hole and the hydrogel material to engage with the hole to seal the hole, thereby limiting subsequent fluid from flowing into or out of the capsule. In this way, the device disclosed herein can utilize the sampling hydrogel as a medium to store microbial samples in the capsule, and can also be used as a device that provides passive mechanical drive to seal the capsule once sampling is completed. In addition, the inventors have understood that after sealing the capsule, the hydrated sampling hydrogel in the capsule can provide an ideal living environment containing nutrients for the sampled bacteria, allowing them to survive before the capsule is recovered. Additionally, according to some aspects, the sealing of the capsule can help protect the sample collected within the capsule from the harsh environment located within the gastrointestinal tract, thereby protecting the bacterial sample stored in the sampling hydrogel sample for subsequent analysis.

[0065] In some embodiments, the capsule may include a biodegradable coating, which includes a plurality of biodegradable coating layers arranged in the sampling hole. For example, the biodegradable coating may be an enteric coating, which is configured to dissolve at the desired target location along the length of the gastrointestinal tract. In one embodiment, an external biodegradable coating layer and an internal biodegradable coating layer are provided in the sampling hole. Therefore, when the capsule reaches the position before the target location (referred to herein as the initial position), the external biodegradable coating layer is soluble, and when the capsule reaches the target location, the internal biodegradable coating layer is soluble. The dissolution of the internal biodegradable coating can allow intestinal fluid to enter the capsule cavity through the hole. In some embodiments, the biodegradable coating can be only arranged in the sampling hole, so that the biodegradable coating is constrained in the sampling hole (and does not extend to the outer surface of the capsule). It should be understood that, as will be understood by those skilled in the art, the capsule according to the present disclosure may include any suitable biodegradable coating. For example, suitable coating materials include, but are not limited to, pH-sensitive polymeric materials such as alkaline butylated methacrylate (EUDRAGIT EPO), polymethacrylic acid-co-ethyl acrylate (EUDRAGIT L100-55), polymethacrylic acid-co-methyl methacrylate (EUDRAGIT L 100), hydroxypropyl methylcellulose phthalate (HP-55), hydroxypropyl methylcellulose phthalate (HPMCP), cellulose acetate phthalate (CAP), and polyvinyl acetate phthalate (PVAP).

[0066] In some embodiments, a biodegradable coating is formed using a solution casting technique. In such a technique, a polymer is dissolved in a solution, and the solution is applied to a cap. In some embodiments, the cap of a sampling capsule is placed on a silicone (e.g., PDMS) holder, which blocks the hole of the cap, while a biodegradable coating is deposited on the cap to prevent the biodegradable coating from passing through the hole. The solution may have a high viscosity and may dry quickly. After the coating is dried, the cap may be removed from the holder. The solution may be easily separated from the silicone holder, so that the coating remains on the cap and covers the hole of the cap. In some embodiments, the coating may be formed by drop casting, which includes releasing large droplets with controlled size and momentum, which diffuse and wet the surface when impacted, as described below. In some embodiments, a doctor blade may be used to produce a film with uniform thickness. A doctor blade, also referred to as a knife coating or a blade coating, involves operating a blade on the surface (or moving the surface below the blade). The small gap between the blade and the substrate determines how much solution can pass through as the blade passes, thus evenly distributing the solution on the substrate.

[0067] In some embodiments, each layer of a biodegradable coating is formed using a drop casting technique. In such techniques, a biodegradable coating polymer is dissolved in a solvent, and the resulting solution is deposited in a sampling hole of a capsule. In some embodiments, the cap of a sampling capsule is placed on a silicone (e.g., PDMS) support, which blocks the hole of the cap, while a first solution containing a first biodegradable coating polymer is deposited into the hole to prevent the first solution from passing through the hole. A droplet of the first solution with a known size can be deposited into the hole with controlled momentum to form a first coating layer with a first thickness. In some embodiments, a second solution containing a second biodegradable coating polymer is deposited into the hole. A droplet of the second solution with a known size can be deposited into the hole with controlled momentum to form a second coating layer with a second thickness.

[0068] In some embodiments, a doctor blade can be used to produce a film with uniform thickness. Doctor blade, also known as knife coating or blade coating, involves operating a blade over a surface (or moving the surface under a blade). The small gap between the blade and the substrate determines how much solution can pass through as the blade passes, thereby evenly distributing the solution on the substrate.

[0069] The thickness of the first layer and the second layer can be the same or different. The combined thickness of the entire coating can be selected to be sufficient to prevent premature rupture of the coating. Selective variation in the thickness of each layer can allow targeted sampling at a specific point through the gastrointestinal tract. 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 can be designed to target a portion of the colon for sampling by selecting the thickness of the second coating that produces a dissolution time that corresponds to the time through the target portion.

[0070] After the coating is dried, the cap can be removed from the stent. The coating can be easily separated from the silicone stent, allowing the coating to remain undamaged in the holes of the cap. In some embodiments, NIR can be used to accelerate and improve the drying process. In the NIR process, an emission wavelength with a higher absorbance in the solvent than in the polymer can be selected to facilitate the removal of the solvent and the drying of the solution. In some embodiments, NIR can be used multiple times in the drop casting process. For example, NIR can be used for the first time to dry the first solution before depositing the second solution, and for the second time to dry the second solution. In the NIR process, an emission wavelength with a higher absorbance in the solvent than in the polymer can be selected to facilitate the removal of the solvent and the drying of the solution. In some embodiments, NIR is used multiple times in the drop casting process. For example, NIR can be used for the first time to dry the first solution before depositing the second solution, and NIR can be used for the second time to dry the second solution.

[0071] As described above, the capsule may include a sealing member, and after the sampling hydrogel absorbs the intestinal fluid sample and expands in the capsule cavity, the sampling hydrogel can press the sealing member to engage with the sampling hole of the capsule. According to some aspects, the sealing member can be configured to provide the required gas permeability between the capsule cavity and the external environment of the capsule. For example, in some embodiments, the sealing member can be formed as a polydimethylsiloxane (PDMS) film, which can provide gas permeability to allow natural gas exchange between the gastrointestinal tract and the inside of the capsule (i.e., the capsule cavity), which can help maintain the natural metabolism of the sampling bacteria, and after the capsule is sealed, promote their survival. Other suitable materials for sealing members include, but are not limited to, polyvinyl chloride (PVC), thermoplastic polyurethane (TPU), cyclic olefin copolymer (COC) and perfluoropolyether (PFPE).

[0072] Depending on the particular embodiment, the capsules described herein may be able to maintain viable bacteria alive for an extended period of time after the capsules are sealed by the sealing member for subsequent analysis. For example, in some embodiments, the sampling hydrogel and / or the sealing member may be constructed and arranged to maintain viable bacteria in the sample fluid alive (i.e., keep the viable bacteria alive) 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 before the sampled fluid is retrieved for analysis.

[0073] In some embodiments, the device can be used to collect proteins or other biomarkers to study physical conditions. For example, in some embodiments, the device can be used to collect calprotectin in the gastrointestinal tract to diagnose inflammatory bowel disease (IBD). Calprotectin is a protein released by neutrophils when the gastrointestinal tract is inflamed.

[0074] After passing through the gastrointestinal tract, the patient can discharge the sealed capsule and then recycle it to analyze the intestinal microbial sample contained therein. According to some aspects, the capsule can be configured and arranged to ensure easy disassembly once it is recovered, so that the sampling hydrogel containing the sample is easily recovered. For example, in some embodiments, the capsule shell can be formed by two or more shell parts, and the two or more shell parts can be movably fixed to each other to allow entry into the capsule cavity. In an exemplary embodiment, the capsule shell can be formed by two capsule parts, and the two capsule parts are connected to each other by a spiral interface, which can make the capsule easy to disassemble after being taken out by excretion, so that the sampling hydrogel in the capsule can be removed for future cultivation and analysis of the bacterial sample contained therein. Other suitable interfaces include but are not limited to snap-fit ​​interfaces and friction or interference fit (interference fit) interfaces.

[0075] According to some aspects, prior to assembling the various components of the capsule shell, the components may be treated to provide a hydrophilic coating on the capsule shell. The inventors have recognized and appreciated that such treatments may help the sample fluid flow through the sampling holes and enter the interior of the capsule cavity, where the sample fluid may be absorbed by the hydrogel material contained therein. In particular, the hydrophilic coating on the surface of the capsule shell may help provide continuous pulling of the fluid from the intestine to the narrow sampling holes on the capsule. In addition, the hydrophilic coating on the inner surface of the sampling holes may help the biodegradable coating adhere to the sampling holes in addition to further helping to pull the liquid from the intestine. For example, in some embodiments, the hydrophilic surface modification may be accomplished by activating the surface of the shell components using an air plasma treatment followed by polyethylene glycol (PEG) treatment.

[0076] The hydrogel materials disclosed herein can be composed of a hydrophilic polymer network that can absorb large amounts of water while maintaining its structure. These polymer networks are typically cross-linked by covalent bonds, hydrogen bonds, van der Waals interactions, and / or physical entanglements. The devices disclosed herein utilize the absorption capacity, as well as the mechanical properties of the hydrogel, to provide a non-invasive sampling device that can passively extract and fix samples from a target location in the gastrointestinal tract.

[0077] In some embodiments, the sampling hydrogel in the capsule can be synthesized by a combination of acrylic acid (AA) and acrylamide (AM) monomers. It should be understood that the present disclosure is not limited to any specific ratio of these monomers to form a hydrogel material. For example, suitable 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 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. Although certain hydrogel materials are described herein, it should be understood that other hydrogel materials may be suitable, such as hydrogels based on other acrylic polymers (e.g., a combination of acrylic acid, acrylamide, poly (N-isopropylacrylamide) and / or poly (N, N-diethylacrylamide)) and / or non-acrylic polymers. In some embodiments, hydrogels can be synthesized from a combination of acrylamide and N,N'-methylenebisacrylamide (MBA).

[0078] It should be understood that the capsule shell according to the present disclosure can be prepared by any suitable biocompatible material. For example, in some embodiments, the capsule shell can be formed by a biocompatible polymeric 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 polymeric materials treated with PEG to provide biocompatibility. In addition, it should be understood that the present disclosure is not limited to any particular method for forming the capsule shell. For example, some embodiments described in more detail below utilize capsule shells formed by a 3D printing process. Other suitable preparation methods may include but are not limited to casting methods, molding methods (such as injection molding) or other methods that can be understood by those of ordinary skill in the art.

[0079] Depending on the particular embodiment, the capsule can have any suitable size. For example, a cylindrical capsule can 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 can have a diameter of about 9 mm and a length of about 15 mm, which is smaller than a standard No. 000 gelatin capsule (which has a size of 9.97×26.14 mm). In addition, the sampling hole formed in the capsule shell can have a diameter selected based on the size of the sealing member contained in the capsule. For example, in some embodiments, the diameter of the sampling hole can be selected to be at least 1 mm smaller than the diameter of the sealing member, which may help ensure a suitable seal between the capsule and the sealing member. In an exemplary embodiment, the sampling hole can have a diameter of about 5 mm.

[0080] With respect to the accompanying drawings, particular non-limiting embodiments are described in further detail. It should be understood that the various systems, components, features, and methods described with respect to these embodiments may be used alone and / or in any desired combination, as the present disclosure is not limited to the particular embodiments described herein.

[0081] Figure 1 is a schematic diagram of a sampling capsule 102 at different locations 10 , 20 , 30 , 40 within the gastrointestinal tract 100 . Figure 2A , 2B 2C and 2D are schematic diagrams of the sampling capsule 102 in various operating states, which correspond to Figure 1 Positions 10, 20, 30, 40 in the table. Figure 2AAs shown, the capsule 102 includes a biodegradable coating 106 (e.g., an enteric coating) having an outer biodegradable coating layer 108 and an inner biodegradable coating layer 110. The biodegradable coating 106 is disposed within a sampling hole 112 of the capsule 102 such that the biodegradable coating 106 seals the sampling hole 112. In this manner, 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 a target location within the gastrointestinal tract. In some embodiments, the biodegradable coating 106 may be disposed only within the sampling hole and not on the outer surface of the capsule. Figure 2B As shown, once the capsule reaches the initial location in the gastrointestinal tract (which may be a point before the target location in the gastrointestinal tract), the outer biodegradable coating layer 108 degrades and exposes the inner biodegradable coating layer 110 to the gastrointestinal fluid surrounding the capsule. Figure 2C As shown, once the capsule 102 reaches the target location, the inner biodegradable coating layer 110 degrades and allows GI fluid containing sample material 118 (e.g., bacteria, proteins, chemicals, and / or potentially other compounds of interest) to enter the capsule, where the fluid is absorbed by the sampling hydrogel 114. Upon absorbing the fluid, the sampling hydrogel swells and expands within the capsule cavity 104 and substantially fills the entire volume of the capsule cavity. In addition, the expansion of the sampling hydrogel presses the sealing member 116 into engagement with the sampling hole, thereby sealing the sampling hole through the mechanical force exerted by the swollen hydrogel, as shown in FIG. Figure 2D As shown in the figure, the fluid absorbed by the sampling hydrogel can contain sample material 118 from the gastrointestinal tract. After the capsule is excreted, the capsule can be disassembled to remove the sampling hydrogel and the bacterial sample contained therein so that the bacteria can be cultured and analyzed in the future.

[0082] When a patient swallows an exemplary embodiment sampling device having two layers of biodegradable polymer coating, the device can be exposed to saliva at pH 7 for typically less than 1 minute. As the device moves toward the stomach, the pH drops to about 3. The device can remain inactive because the external enteric polymer remains non-ionized in the acidic pH environment of the stomach. As the device moves further down the gastrointestinal tract, when it reaches the small intestine, the pH increases, with an average pH of 6.8 and a typical maximum pH of 7.5. When the pH exceeds the dissolution threshold of the external biodegradable coating layer, the external polymer begins to ionize, initiating polymer dissolution. Once the external biodegradable coating layer dissolves, the internal biodegradable coating layer is exposed to the fluid of the small intestine. At this point, the device can remain inactive because the internal biodegradable coating layer remains non-ionized in the alkaline pH environment of the small intestine. When the device reaches the ascending colon, the pH suddenly drops again, and the internal biodegradable coating layer may begin to ionize and dissolve in the acidic environment of the colon. Once the inner biodegradable coating layer dissolves, intestinal fluid can enter the capsule cavity 104, and the hydrogel can begin to swell by absorbing the intestinal fluid while pushing the sealing member in the capsule cavity toward the sampling hole. When the device hole is sealed with the sealing member, the device can move through the rest of the gastrointestinal tract without fluid exchange.

[0083] In some cases, the device may need about 24 to 72 hours to pass through the colon.Depending on the intended application of the device, the biodegradable coating dissolution and / or hydrogel extension can be adjusted to the desired target position in the colon.The dissolution of the biodegradable coating can be adjusted by selecting the appropriate thickness of one or more coating layers as described above.For example, in some embodiments, the biodegradable coating can be configured to target the ascending colon by selecting the coating thickness of the internal biodegradable coating layer to produce complete dissolution within two hours after the device enters the colon.Then, the hydrogel can be configured to extend within one hour, and finally, after this, the device can be configured to be sealed by a sealing member within 0.5 hours.In the example of targeting the transverse colon, the biodegradable coating can be configured to target the ascending colon by selecting the coating thickness of the internal biodegradable coating layer to produce complete dissolution within 11 hours after the device enters the colon, the hydrogel can be configured to extend within one hour, and finally, after this, the device can be configured to be sealed by a sealing member within 0.5 hours.These time target time intervals can be selected to allow a safety margin (margin) of the desired time length to ensure that the device is sealed before excretion. After voiding, the capsule can be removed, disassembled, and samples can be analyzed for further studies.

[0084] Figure 32 is an exploded schematic diagram of an embodiment of a capsule 202 for passive sampling of the gastrointestinal tract. The capsule 202 includes a capsule shell 220, which includes a first shell portion 222 (also referred to herein as a cap) and a second shell portion 224. The first shell portion includes a sampling hole 212 to provide fluid communication between the outside of the capsule and the capsule cavity 204 formed in the capsule shell 220. A biodegradable coating 206 including an outer biodegradable coating layer 208 and an inner biodegradable coating layer 210 is provided in the sampling hole 212 to seal the sampling hole 212 until the capsule 202 reaches a desired location in the gastrointestinal tract. In some embodiments, the biodegradable coating may be provided only in the sampling hole, so that the biodegradable coating 206 is constrained in the sampling hole (and does not extend to the outer surface of the capsule). The sampling hydrogel 214 and the sealing member 216 are located in the capsule cavity 204, and as shown, the sealing member 216 is located between the sampling hydrogel 214 and the sampling hole 212. As described above, in some embodiments, the sealing member 216 can be a flexible membrane, such as a PDMS membrane, and can provide gas permeation between the capsule cavity 204 and the outside of the capsule 202 after the sealing member seals the capsule cavity. In addition, the first and second housing parts 222 and 224 include a connection interface 226 to allow the capsule housing to be easily disassembled after collecting the sample to remove the hydrogel material. In the described embodiment, the connection interface 226 includes corresponding thread features formed on the first and second housing parts, which are combined to form a spiral interface. However, as described above, in other embodiments, other connection arrangements such as snap fit or interference fit may be suitable.

[0085] In the described embodiment, the sampling hole has a circular shape. However, it should be understood that the sampling hole 212 can have any suitable shape. The shape of the sampling hole 212 can determine the shape of the biodegradable coating 206 disposed therein. For example, in some embodiments, the sampling hole can be square, rectangular, oval, or any other suitable shape.

[0086] Figures 4A-4F A schematic diagram of one embodiment of a method of making a capsule having a multi-layer biodegradable coating disposed within a sampling hole of the capsule is shown. Figure 4A, the first shell portion 322 of the capsule is placed on a fixture 330 in the direction of the arrow shown. The fixture 330 has a sealing plate 332, which is configured to provide a liquid-tight seal with the first shell portion 322 of the capsule at the periphery of the sampling hole 318 of the capsule. The sealing plate 332 can be a silicone material (e.g., PDMS) or any other material that provides a liquid-tight seal without bonding with or adhering to the biodegradable coating. A liquid solution of the first biodegradable coating 308 is introduced into the hole 318. The liquid-tight seal between the sealing plate 332 and the first shell portion 322 of the capsule can retain the first biodegradable coating 308 in the hole, as shown. Figure 4C The first biodegradable coating 308 is then dried. In some embodiments, drying may include using NIR, such as Figure 4C Any suitable parameters for NIR, including wavelength, operating power, and scanning speed, may be appropriately selected, as described below.

[0087] In some embodiments, the wavelength used in the NIR drying process can be greater than or equal to 300mm, 400nm, 500mm and / or any other suitable wavelength. In some embodiments, the wavelength can be less than or equal to 2.0μm, 2.25μm, 2.5μm and / or any other suitable wavelength. Consider the aforementioned combination, including, for example, greater than or equal to 300mm and less than or equal to 2.5μm, greater than or equal to 400mm and less than or equal to 2.25μm, and / or any other suitable combination of the aforementioned. Of course, although a specific range of NIR wavelengths is provided above, it should be understood that other ranges greater than and less than the ranges indicated above are also considered, because the present disclosure is not limited in this way.

[0088] In some embodiments, the operating power used in the NIR drying process can be greater than or equal to 1kW, 3kW, 5kW and / or any other suitable power level. In some embodiments, the operating power can be less than or equal to 8kW, 10kW, 15kW and / or any other suitable power level. Consider the combination of the foregoing, including, for example, being greater than or equal to 1kW and less than or equal to 15kW, being greater than or equal to 3kW and less than or equal to 8kW, and / or any other suitable combination of the foregoing. Of course, although the specific range of NIR operating power is provided above, it should be understood that other ranges greater than and less than the above-noted range are also considered, because the present disclosure is not limited in this way.

[0089] In some embodiments, the scanning speed used in the NIR drying process can be greater than or equal to 1m / min, 3.6m / min, 5m / min and / or any other suitable speed. In some embodiments, the scanning speed can be less than or equal to 7.5m / min, 10m / min, 12.5m / min and / or any other suitable speed. Consider the combination of the aforementioned, including for example being greater than or equal to 1m / min and less than or equal to 12.5m / min, being greater than or equal to 3.6m / min and being less than or equal to 7.5m / min and / or any other suitable combination of the aforementioned. Of course, although the specific range of NIR scanning speed is provided above, it should be understood that other scopes greater than and less than the scope pointed out above are also considered, because the present disclosure is not limited in this way.

[0090] Once the first biodegradable coating 308 has dried, the second biodegradable coating 310 may be introduced into the apertures 318, such as Figure 4D The second biodegradable coating 310 is then dried. In some embodiments, drying may include using NIR, such as Figure 4E 334 in the figure. The first shell 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 can produce a biodegradable coating that is only disposed within the sampling hole, so that the biodegradable coating is constrained within the sampling hole (and does not extend to the outer surface of the capsule).

[0091] Figure 5A schematic diagram of an alternative embodiment of a capsule 402 is shown. The capsule 402 includes a biodegradable coating 406 (e.g., an enteric coating) having an outer biodegradable coating layer 408 and an inner biodegradable coating layer 410. The biodegradable coating 406 is disposed on an outer surface 418 of the capsule 402 and across a sampling hole 412 of the capsule 402, such that the biodegradable coating 406 seals the sampling hole 412. In this manner, the biodegradable coating 406 protects the components within the capsule cavity 404 of the capsule, including the sampling hydrogel 414 and the sealing member 416, and delays sampling until the capsule reaches a target location within the gastrointestinal tract. Once the capsule reaches an initial location in the gastrointestinal tract (which may be a point in the gastrointestinal tract before the target location), the outer biodegradable coating layer 408 degrades and exposes the inner biodegradable coating layer 410 to the GI fluid surrounding the capsule. Once the capsule 402 reaches the target location, the internal biodegradable coating layer 410 degrades and allows the GI fluid containing bacteria to enter the capsule, where the fluid is absorbed by the sampling hydrogel 414. While absorbing the fluid, the sampling hydrogel swells and expands within the capsule cavity 404 and substantially fills the entire volume of the cavity. In addition, the expansion of the sampling hydrogel presses the sealing member 416 to engage with the sampling orifice, thereby sealing the sampling orifice by the mechanical force applied by the swollen hydrogel. The fluid absorbed by the sampling hydrogel can contain bacteria from the gastrointestinal tract. After the capsule is excreted, the capsule can be disassembled to remove the sampling hydrogel and the bacterial sample contained therein so that the bacteria can be cultured and analyzed in the future.

[0092] In the described embodiment, the biodegradable coating 406 initially covers or surrounds only a portion of the capsule 402. However, it should be understood that other arrangements may be suitable, such as arrangements in which the biodegradable coating 406 covers or surrounds the entire exterior of the capsule 402. 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 any shape that seals the sampling hole of the capsule. Figure 5 The biodegradable coating can be deposited on the capsule by any suitable method, including drop casting, doctor blade, solution casting or spraying. It should be noted that any of these methods can produce a biodegradable coating that can be placed only within the sampling hole, so that the biodegradable coating is constrained within the sampling hole (and does not extend to the outer surface of the capsule). Example

[0093] Equipment Manufacturing

[0094] In one embodiment, a device for passive sampling of the colon was prepared using 3D printing. The device consists of four components: a biodegradable coating including a first and a second biodegradable coating layer, 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 using SolidWorks (Dassault Systèmes) and printed using stereolithography using a Form 3 3D printer, followed by rinsing with pure isopropyl alcohol (IPA) and UV curing at 60°C for 15 minutes. The PDMS membrane was prepared using a standard 1:10 ratio of curing agent to silicone base and cured at 70°C for 4 hours. A circle with a diameter of 6 mm was cut in PDMS using a computer-controlled CO2 laser cutting and engraving system (PLS6MW from Universal laser, Inc., operating at a wavelength of 10.6 μm). The hydrogel was synthesized by dissolving acrylamide and MBA crosslinkers in water. The solution was then degassed by bubbling nitrogen for 10 minutes, and then ammonium persulfate (APS) was added to the solution as an initiator. Finally, the solution was poured into a mold and polymerized at 70°C overnight. 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.

[0095] Characterization of biodegradable coatings

[0096] pH sensitive polymers include the broad categories of polyacids and polybases. Polyacids can accept protons at low pH values ​​and release protons at neutral pH values ​​or higher. Therefore, polyacids such as Eudragit L100, Eudragit L100-55, and Eudragit S100 can be dissolved in aqueous media at high pH values, but are insoluble in the lower pH range. Polybases can accept protons at higher pH values ​​and release protons at neutral pH or lower pH ranges. Therefore, polybases such as chitosan and Eudragit EPO can be dissolved in aqueous media at low pH values, but are insoluble in the higher pH range. The release and acceptance of protons, as well as the solubility or insolubility of these polyelectrolytes, are controlled by the pH of the surrounding environment.

[0097] To better evaluate the solubility of various pH-sensitive polymers, methylene blue and dark orange food coloring dyes were added to solutions of Eudragit L100-55 and Eudragit EPO, respectively. These dyes allowed the polymers to reach peak absorption during UV-Vis spectrophotometry (UV-Vis). The solutions were then cast on acrylic plates using a doctor blade to obtain uniform film thickness. After casting each layer, in order to avoid solvent entrapment in the film components, NIR (Adphos NIR-126-250 Modul, the conveyor line speed was 3.6 m.min -1 The films were completely dried using a laser cutting and engraving system at 10.6 μm wavelength to cut identical discs of 10 mm in diameter.

[0098] The dissolution test of pH sensitive polymer samples was carried out in 900mL of various dissolution media, at 37°C using a USPII type instrument (PTWS instrument, Pharma Test, Hainburg, Germany) with a paddle speed of 100rpm. The dissolution medium has a pH value of 1.2, 3, 5.5, 6.8 and 7.4. The polymer sample was taken out from the dissolution medium at a predetermined time point, and before returning to the dissolution medium, a BMG Clariostar microplate reader (BMG Labtech, Germany) was used to analyze by UV-Vis. The percentage of dye released from the formulation was measured using UV-Vis technology at a wavelength corresponding to the peak wavelength of the appropriate dye. All tests were repeated three times.

[0099] Under the gastrointestinal pH environment, the different polyacids obtained ( Figures 6A-6D ) and polybasic alkali ( Fig. 7A -7C) dissolution tests showed various dissolution profiles, indicating that different formulations can achieve different dissolution kinetics and profiles.

[0100] In a polyacid polymer simulating a fasting state of the stomach (pH = 1.2), Figures 6A-6C As shown, all three polyacids showed minimal dissolution (<10%) during the simulated gastric residence time (2 hours). Continuing the experiment up to a residence time of 8 hours did not result in a substantial change in this trend. Figures 6A-6C As shown, all three polyacids showed less than 15% dissolution within 2 hours of gastric residence time. However, Eudragit L100-55 showed a slow, continuous dissolution trend, reaching ~30% after 8 hours of residence time ( Fig. 6A ).

[0101] At pH = 5.5, which typically simulates the pH of the duodenum and cecum, Eudragit L100-55 showed ~30% dissolution after 2 hours ( Fig. 6A However, Eudragit L100 and Eudragit S100 were still mostly insoluble after 8 hours, showing 21% ( Figure 6B ) and 11% ( Figure 6C ) dissolution. When pH = 6.8, which usually simulates the proximal small intestine region, the threshold pH level for Eudragit L100-55 dissolution is reached. This may result in Eudragit L100-55 being completely ionized and completely dissolved within 2 hours ( Fig. 6A Eudragit L100 was completely dissolved after 5 hours ( Figure 6B ), Eudragit S100 only dissolved 20% after 8 hours ( Figure 6C ).

[0102] The dissolution curves of polybasic polymer Eudragit EPO and chitosan are as follows Fig. 7A -7C. Chitosan exhibited relatively rapid dissolution at all pH levels, with complete dissolution occurring within 5-6 hours at each pH level. Eudragit EPO was completely dissolved within 1 hour at pH levels of 1.2, 3.0, and 5.5, and within 3 hours at pH level 6.8. However, at pH 7.4, dissolution of Eudragit EPO did not exceed ~30% over the entire 8-hour residence time.

[0103] Near infrared drying

[0104] In some studies, NIR technology was applied to increase the evaporation rate of the solvent and prevent the mixing of polymer layers while maintaining the integrity of the polymer film structure.

[0105] Characterization of reduced cytotoxicity using NIR

[0106] The in vitro cytotoxicity of samples with a double-layer biodegradable coating was determined by MTT (tetrazolium bromide) assay using human mesenchymal stem cells (hMSCs). The cytotoxicity was studied before and after NIR treatment. The cytotoxicity of capsules without a biodegradable coating was selected as a control, in part to ensure that the cured 3D printed resin was non-toxic to cells. The cytotoxicity of the biodegradable coating was examined because the coating included two drop-cast layers. An examination was performed to verify whether the drop-cast layers released organic solvents that are potential carcinogens and neurotoxins.

[0107] The 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. Once the cells were confluent (~80%), the samples were transferred to a 24-well plate and UV sterilized for 2 hours (1 hour per side) and then plated at 2×10 4 , cells / well density and 2mL final volume were inoculated. After 24 hours of culture, the culture medium in each well was replaced with 1800μL fresh DMEM and 200μL MTT solution (5mg / mL thiazolyl blue dissolved in PBS) and cultured for another hour. The optical density (OD) value of the mixture was measured at 490nm using a BMG microplate reader. Cell viability was calculated using the following equation:

[0108] Cell viability percentage = live cells on the test surface / live cells on the control surface × 100%

[0109] like Figure 8 As shown, the cell viability of the control was calculated to be 92%, indicating that the capsule shell is biosafe. However, the cell viability of the drop-cast film dried in the environment before NIR application was shown to drop to <50%. This toxicity of the bilayer film may be mainly driven by the top layer, because a larger proportion of the solvent may have been trapped within the structure of Eudragit L100-55 due to the higher molecular weight of the solvent compared to the bottom layer with a lower molecular weight, which will be discussed in more detail below.

[0110] The cell viability after NIR heat treatment increased to 84%, indicating that polymer dissolution was not harmful to the cells. The biocompatibility of the NIR treated biodegradable coating was due to the evaporation of the trapped solvent. This is consistent with the thermogravimetric analysis (TGA) results discussed below.

[0111] ATR-FTIR Characterization of NIR Effect

[0112] To ensure that the bilayer drop-cast films showed no intermixing effects after NIR treatment, attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was performed using a PerkinElmer Spectrum 100 FTIR spectrometer at 4000 cm -1 Up to 550cm -1 The IR spectra of the single layers of L100-55 and EPO polymers were compared to the IR spectra of the bilayer films by performing 50 scans over a range of 1000 nm. Single layers of Eudragit L100-55 and Eudragit EPO were evaluated in both the film and powder states. Fig.9A Comparative ATR-FTIR spectra of pure EPO and L100-55 powders are shown, and Fig. 9B Comparative ATR-FTIR spectra of pure EPO and L100-55 films as well as double-layer coated films are shown.

[0113] like Fig.9A As shown in Figure 2, the ATR-FTIR spectra of pure L100-55 and EPO powders are at about 1700 cm -1 and 1100cm -1 The main characteristic peaks are shown at 2850 cm-1 and 2860 cm-2, which correspond to the C=O stretching vibration mode of the ester group and the CO bond of the carboxylic acid ester. -1 There is a stretching band at the position, which may be a dimethylamine group, which can distinguish L100-55 from EPO.

[0114] like Fig. 9B As shown, the ATR-FTIR spectrum of the film shows important peaks for L100-55 and EPO without any obvious additional vibration bands for solvent residues. These observations reveal that NIR treatment is a drying technique capable of removing all solvent without affecting the structure of the polymer film.

[0115] Likewise Fig. 9B As shown, the ATR-FTIR spectrum of the double-layer coating film shows the characteristic peaks of pure L100-55 polymer without any obvious peaks, which indicates that the EPO layer is mixed into the L100-55 film. Since the top coating is a pure L100-55 film, this observation confirms that the expected separation of the two polymer films may originate from the immiscible behavior of the initial polymer solutions.

[0116] TGA Characterization of NIR Effect

[0117] Eudragit L100-55 (Powder and Thin Film Formulations) using Thermogravimetric Analysis (TGA) Fig. 10A ) and Eudragit EPO ( Fig. 10B ). In one film configuration, the film was allowed to dry in the ambient environment. In another film configuration, the film was dried using NIR. No solvent was used in the powder configuration.

[0118] The difference in weight loss between powder and film constructions may be due to the loss of water and solvent present in the film construction, as these factors are not present in the powder construction. In addition, films dried in ambient conditions may entrap a higher proportion of solvent than films dried with NIR. Fig. 10A and 10BAs shown, NIR drying removes a greater proportion of the entrapped solvent than ambient drying, resulting in a TGA curve that is closer to that of the powder form.

[0119] In all cases of Eudragit L100-55 ( Fig. 10A ), the weight loss at 50-150°C may be due to the evaporation of excess solvent, while the weight loss at 225-425°C may be due to thermal degradation of the polymer structure. Fig. 10B For Eudragit EPO in , the loss at 275-325 °C is likely the result of solvent evaporation, while the larger drop at 375-450 °C is likely due to thermal degradation.

[0120] The effect of NIR on Eudragit L100-55 was significantly greater than that on Eudragit EPO. This may be due to the higher molecular weight of Eudragit L100-55. Eudragit L100-55, with a molecular weight of 320,000 g / mol, is heavier and stickier than Eudragit EPO, with a molecular weight of only 47,000 g / mol. This higher molecular weight may lead to a significant increase in the entanglement and embedding of solvent particles, which may make the solvent particles more difficult to remove without the help of drying techniques such as NIR. As a result, it was observed that the difference between Eudragit L100-55 dried by NIR and Eudragit L100-55 dried in ambient environment was significantly greater than the difference between Eudragit EPO dried by NIR and Eudragit EPO dried in ambient environment. Because the radiation from the NIR is not destructive to the polymer structure, the solvent molecules can be targeted for evaporation without affecting the film structure or the capsule body.

[0121] Microscopic characterization of NIR effects

[0122] To visualize the effect of NIR on the morphology and mixing of the bilayer biodegradable coating, cross-sectional images of the bilayer coating were taken. Since the solvents of Eudragit L100-55 and Eudragit EPO are essentially the same, the polymers are likely to mix if the layers are slowly dried under ambient conditions. In some cases, mixed polymers may produce unexpected dissolution profiles at various pH levels. For example, Fig.11A The bilayer formation is shown when the top and bottom layers are dried in ambient environment. Fig.11AThe mixture shown demonstrates that the two polymers are blended with each other. In this configuration, the bilayer coating may not exhibit the expected response to various pH levels. Mixing of the layers may have occurred in part because the slow drying process of the top layer may have allowed the solvent to dissolve the bottom layer. This effect, where the application of the top layer can cause the dissolution of the bottom layer over time, can lead to mixing of the polymers.

[0123] Fig. 11B Shown after 2 hours exposure to a pH level of 1.2 Fig.11A Under these conditions, the top layer could not protect the bottom layer from dissolution, probably due to the mixing of the two layers. Therefore, the orange layer was completely dissolved, and the thickness of the blue layer was significantly reduced. Fig. 11C It is shown that after 6 hours at pH 6.8, the remaining top layer is completely dissolved. This dissolution of the top layer should be expected since the pH level exceeds the pH dissolution threshold of the top layer polymer (Eudragit L100-55). Fig.11D It is simply shown that the bottom layer has dissolved due to mixing, so at a pH level of 5.5, no bottom layer can be dissolved.

[0124] Figures 11E-11H The effect of NIR treatment on the double layer coating is shown. NIR treatment is used to prevent polymer mixing by drying the film quickly and efficiently. Fig.11E As shown, the bottom and top layers are more clearly separated and better separated into individual layers. After immersion in pH 1.2 for 2 hours, the layers showed little dissolution or thickness change ( Fig.11F ). This is probably because the pH did not exceed the dissolution threshold of the Eudragit L100-55 top layer, so the top layer could be preserved and act as a shield to prevent the bottom layer from being exposed to a lower pH environment, which would otherwise cause the bottom layer to dissolve. However, when the pH was raised to 6.8, as Fig.11G As shown, the dissolution threshold of the top layer is met. Fig.11G The top layer was completely dissolved after 6 hours at a pH level of 6.8. Once the top layer was dissolved, the bottom layer was exposed to a higher pH environment. The pH was then lowered to 5.5 for 1 hour, and the bottom layer was completely ionized and dissolved, as shown in FIG. Fig.11H shown.

[0125] SEM Characterization of NIR Effect

[0126] also, Fig. 12A and 12B High magnification cross-sectional images of the double-layer biodegradable coating before and after NIR treatment are shown in FIG. These images were taken using a scanning electron microscope (SEM) (Hitachi-S 4800, Tokyo, Japan). Fig. 12AAs shown, the top layer (Eudragit L100-55, dried at ambient) exhibits significant porosity. The bottom layer (Eudragit EPO, dried at ambient) has significantly lower porosity than L100-55, which may be formed by slow drying under ambient conditions. In addition, as described above, the higher molecular weight of Eudragit L100-55 may cause a greater degree of entanglement between polymer chains and solvent and air particles, effectively preventing solvent particles and air particles from easily escaping from the polymer structure.

[0127] Fig. 12B shows that when NIR is used to dry the coating layer, there is no similar porosity issue in the Eudragit L100-55 top layer. This is probably due to the pulsed heat from the lamp that excites the solvent particles and air particles, thus forcing the air bubbles to escape the polymer matrix, ultimately resulting in a pore-free and crack-free polymer structure. In short, the microscopy and SEM cross-sectional images after NIR treatment can prove that this technology not only achieves proper dissolution and protection in different pH environments, but also helps to obtain acceptable polymer morphology.

[0128] Bacterial sampling

[0129] In vitro studies

[0130] To evaluate the sampling capabilities of the hydrogel and the entire device, four containers containing a mixture of intestinal microbiota were prepared. Each container contained a different mixture, as shown in Table 1 below. The composition of the samples drawn by each device and each hydrogel was compared with 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 the corresponding broth in an anaerobic chamber, as also shown in Table 2.

[0131]

[0132]

[0133]

[0134] 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 entire device was disassembled. The disassembled components and the free-swelling hydrogel were transferred to separate vials with 4 mL of deionized water and extracted on a stirrer at 100 rpm for 2 hours.

[0135] After 2 h of extraction, the TM PureLink TM DNA extraction was performed using the Genomic DNA Mini Kit (Invitrogen, USA) on extraction buffer (DI water). Each bacterial mixture was aliquoted with the device or hydrogel before sampling ("Pro") and after sampling ("Post"). Table 1 shows that all bacterial compositions sampled with the complete device were similar to the corresponding mixtures (Pro and Post). Although the results using the hydrogel alone are less representative, this in vitro bacterial sampling experiment confirms the ability of the capsule device to extract representative samples of the intestinal microbiota.

[0136] In vivo studies

[0137] In vivo evaluation of device functionality was performed using pigs. The experiment was approved by the Purdue University Animal Care and Use Committee under protocol number 1911001975A002. Prior to the experiment, the pigs were weighed (each weighing approximately 40-50 kg) and housed individually in metabolic boxes. The pigs had a 5-day acclimatization period in the new environment and had free access to water. Their diet was formulated based on corn and soybean meal to meet or exceed NRC (2012) requirements. Pigs were fed a daily level of 3 times the estimated energy maintenance requirement (i.e., 197 kcal ME / kg BW0.60; approximately 4% of the pig's body weight per day), with daily feed rations provided every morning.

[0138] Prior to capsule administration, the pigs were fasted for 12 hours. Two hours after capsule administration, the pigs were fed a diet containing the indigestible iron oxide marker to determine the digestive passage rate. The pigs were then observed at 30-minute intervals to determine the length of time required for the marker to be excreted into the feces (red).

[0139] The capsules were administered orally using a balling gun designed for use with pigs. Once the capsules were expelled and collected, each pig was euthanized to measure the pH of various parts of the gastrointestinal tract. To avoid potential errors in fluid homogenization and pH values, zip ties were used to isolate the stomach, small intestine, and large intestine. For more accurate analysis, small intestine sections (e.g., 2-meter increments) were cut. Fig.13A , 13B Duo: duodenum, J: jejunum, Ile: ileum) used in the study, whereas the large intestine was sliced ​​in 1-meter increments (e.g. Fig.13A ,13B LI used in: large intestine). The fluid contents of each section were poured into a disposable plastic weighing boat, and the pH value was obtained from these fluid contents.

[0140] The administered capsules were retrieved from the animals after defecation. After wiping and rinsing the external excreta, the capsules were disassembled. The decomposed components were transferred to separate vials with 4 mL of sterile DI water and extracted on a stirrer at 100 rpm for 2 hours.

[0141] 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 was measured using a NanoPhotometer NP60 (Implen, Germany).

[0142] The V4 region of the 16S rRNA gene was amplified using PCR primers: 515 forward, 5'GTGCCAGCMGCCGCGGTAA, 806 reverse, 5'GGACTACHVGGGTWTCTAAT, for all DNA extraction samples. TM PCR reactions were performed individually in a total volume of 50 μL using Phusion High-Fidelity PCR Master Mix (Thermo Scientific, USA) according to the manufacturer's recommendations. The expected amplicon size (~254 bp) was verified by 2% agarose gel electrophoresis. TM PureLink TM The PCR product was purified using PCR purification kit (Invitrogen Life Technologies, USA).

[0143] After checking sample quality using NanoPhotometer NP60, PCR products were sent to Purdue Genomics Core Facility for WideSeq (next generation sequencing) analysis. Nextera DNA library preparation kit (Illumina, CA, USA) was used to create sequencing libraries, and multiple libraries were sequenced using MiSeq (Illumina, CA, USA) to produce paired end reads (read) (2x250bp). Paired end reads from each sample were processed to remove adapters and bases with poor quality. Only the filtered paired reads were used for further processing. Then, reads were reconstructed into full sequences, and taxonomic assignments were performed using the Silva rRNA database.

[0144] The V4 region of 16S rRNA was amplified and sequenced from 17 different cavity content samples from Pig 1 and Pig 2. The average value of 52,413 reads per sample was obtained by MiSeq instrument (Illumina, CA, USA). The sequence was processed to remove adapters and bases of poor quality. The filtered reads were reconstructed into full sequences and taxonomically assigned using the Silva rRNA database, which was summarized into 12 gates. The results were used to create the microbiological composition profiles of Pig 1 and Pig 2.

[0145] Fig.13A and 13B The results shown in show that the microbiota composition can vary significantly along the pig intestine, especially between the small intestine and the large intestine. The most dominant phylum throughout the intestine is Firmicutes (64%-99%). The Bacteroidetes phylum is increased in the large intestine sections, accounting for 19%-32% of the total bacteria. The difference between the Bacteroidetes / Firmicutes ratio in the small intestine and the Bacteroidetes / Firmicutes ratio in the large intestine can be used as a biomarker to indicate the sampling location of the device.

[0146] The two devices administered to Pig 1 and Pig 2 were also collected and analyzed by WideSeq using the same method. Capsule 1 from Pig 1 was found in the feces of Pig 1. Capsule 2 from Pig 2 was not expelled at the time of euthanasia and was found in the large intestine sample when the gastrointestinal tract was dissected. The Firmicutes and Bacteroidetes compositions of Capsule 1 were 73% and 24%, respectively. This result was very similar to the large intestine 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 intestine sections of Pig 2 (0.02-0.06%). This indicates that the sampling device did indeed extract samples from the large intestine as planned and expected.

[0147] Although the teaching of the present invention has been described in conjunction with various embodiments and examples, it is not intended that the teaching of the present invention be limited to the embodiments or examples. On the contrary, the teaching of the present invention includes multiple substitutions, modifications and equivalents, as will be appreciated by those skilled in the art. Therefore, the above description and the accompanying drawings are only examples.

Claims

1. A passive sampling device for the gastrointestinal tract, the device comprising: A capsule shell surrounding the capsule cavity; a sampling hole formed in the capsule shell and providing fluid communication between the capsule cavity and the exterior of the capsule shell; a sampling hydrogel located within the vesicle cavity, wherein when exposed to the sample fluid, the sampling hydrogel is configured to absorb the sample fluid, swell within the vesicle cavity, and store the sample fluid for subsequent analysis; a sealing member located in the capsule cavity between the sampling hydrogel and the sampling hole, wherein expansion of the sampling hydrogel in the capsule cavity presses the sealing member into engagement with the sampling hole to seal the capsule cavity; and A biodegradable coating covers the sampling hole, the biodegradable coating comprising a plurality of biodegradable coating layers, wherein degradation of the plurality of biodegradable coating layers exposes the sampling hole to allow fluid to flow into the capsule cavity.

2. The device of claim 1, wherein a plurality of biodegradable coating layers are disposed within the sampling hole.

3. The device of claim 2, wherein a plurality of biodegradable coating layers are confined within the sampling aperture.

4. The device of claim 1, wherein a plurality of biodegradable coating layers are disposed on the outer surface of the capsule and across the sampling aperture.

5. The device of any one of claims 1-4, wherein the plurality of biodegradable coating layers comprises a first biodegradable coating layer and a second biodegradable coating layer.

6. The device of claim 5, wherein the first biodegradable coating layer comprises a first biodegradable coating layer configured to degrade at a first predetermined location in the gastrointestinal tract, and the second biodegradable coating layer comprises a second biodegradable coating layer configured to degrade at a second predetermined location in the gastrointestinal tract different from the first predetermined location.

7. The device of claim 6, wherein the first biodegradable coating layer is configured to degrade in the small intestine of the gastrointestinal tract and the second biodegradable coating layer is configured to degrade in the large intestine of the gastrointestinal tract.

8. The device of claim 5, wherein the first biodegradable coating layer is configured to degrade when exposed to a first pH level, and the second biodegradable coating layer is configured to degrade when exposed to a second pH level different from the first pH level.

9. The device of claim 8, wherein the first biodegradable coating layer is configured to degrade at a pH greater than 5.5, and the second biodegradable coating layer is configured to degrade at a pH less than 5.

5.

10. The device of claim 1, wherein the capsule shell comprises a hydrophilic coating.

11. The device of claim 10, wherein the hydrophilic coating is formed by exposing the capsule shell to a plasma treatment followed by a polyethylene glycol treatment.

12. The device of claim 1, wherein the sampling hydrogel is synthesized from acrylamide monomers.

13. The device of claim 1, wherein the sampling hydrogel is synthesized from acrylic acid and acrylamide monomers.

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

15. The device of claim 1, wherein the sealing member is at least partially gas permeable.

16. The device of claim 15, wherein the sealing member is a polydimethylsiloxane membrane.

17. The device of claim 1, wherein the capsule housing comprises a first housing portion and a second housing portion that are removably connected to each other to form the capsule housing.

18. The device of claim 17, wherein the first and second housing portions are connectable to each other via a threaded interface.

19. The device of claim 17, wherein the sampling aperture is formed in the first housing portion.

20. The device of claim 1, wherein the sampling hydrogel is constructed and arranged to maintain viable bacteria present in the sample fluid for at least 1 hour after sealing the capsule cavity.

21. The device of claim 20, wherein the sampling hydrogel is constructed and arranged to maintain viable bacteria present in the sample fluid for up to 24 hours after sealing the capsule cavity.

22. The device of claim 1, wherein the sampling hydrogel is the only component within the capsule housing configured to absorb sample fluid.

23. The device of claim 1, wherein the sealing member is formed by solution casting.

24. The device of claim 1, wherein the biodegradable coating is formed by drop casting.

25. The device of claim 1, wherein the biodegradable coating is formed by drop casting.

26. A method of making a device for passive sampling of the gastrointestinal tract, the method comprising: providing a capsule housing having a capsule cavity and a sampling hole, the sampling hole providing fluid communication between the capsule cavity and the exterior of the capsule housing; as well as The sampling hole is closed with a biodegradable coating comprising a plurality of biodegradable coating layers, wherein degradation of the plurality of biodegradable coating layers exposes the sampling hole to allow fluid to flow into the capsule cavity.

27. The method of claim 26, wherein closing the sampling hole with a plurality of biodegradable coatings comprises depositing a plurality of biodegradable coating layers within the sampling hole using a drop casting technique.

28. The method of claim 27, wherein using the drop casting technique comprises placing a first portion of a capsule shell on a sealing plate, the first portion of the capsule shell comprising a sampling hole, and the sealing plate is configured to provide a liquid-tight seal with the sampling hole, depositing a plurality of biodegradable coating layers within the sampling hole using the drop casting technique, and removing the first portion of the capsule shell from the sealing plate.

29. The method of claim 26, further comprising drying the plurality of biodegradable coating layers using near infrared radiation.

30. The method of claim 26, wherein the biodegradable coating is disposed on only a portion of the capsule shell.

31. The method of claim 26, wherein the biodegradable coating is disposed within the sampling well.

32. The method of claim 31, wherein the plurality of biodegradable coating layers of the biodegradable coating are confined within a sampling hole.

33. The method of claim 26, wherein the biodegradable coating is formed into a flat surface.

34. The method of claim 26, further comprising providing a sampling hydrogel within the vesicle cavity, wherein when exposed to the sample fluid, the sampling hydrogel is configured to absorb the sample fluid, swell within the vesicle cavity and store the sample fluid for subsequent analysis.

35. The method of claim 26, wherein closing the sampling hole with a biodegradable coating comprising a plurality of biodegradable coating layers comprises closing the sampling hole with a first biodegradable coating layer, drying the first biodegradable coating layer using near infrared radiation, depositing a second biodegradable coating layer onto the first biodegradable coating layer, and drying the second biodegradable coating layer using near infrared radiation.

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