Thermo-responsive probiotic vesicles for IBD treatment

Abstract

The present disclosure introduces a sprayable formulation for the enhanced treatment of inflammatory bowel disease (IBD). A lyophilized formulation (102) comprises probiotic- derived extracellular vesicles (EVs) (104) sourced from the VSL#3 probiotic blend, encapsulated within a thermo-responsive polymer matrix (106) made from poly(N-isopropyl acrylamide) (PNIPAM) and hyaluronic acid. Available as an off-the-shelf lyophilized powder, the formulation (102) is easy to store and rehydrate for use. Upon application, the lyophilized formulation (102) undergoes a phase transition at physiological temperature, forming a gel-like coating over the inflamed areas of the gut. This matrix (106) ensures localized delivery of the encapsulated EVs (104) directly to the disease site. The combination of hyaluronic acid and probiotic-derived EVs (104) reduces inflammation and promotes mucosal healing, addressing critical aspects of gut health in IBD management.

Description

THERMO-RESPONSIVE PROBIOTIC VESICLES FOR IBD TREATMENTTECHNICAL FIELD

[0001] The present disclosure pertains to the field of pharmaceutical formulations and treatments. Moreover, it involves a sprayable therapeutic composition for the enhanced treatment of inflammatory bowel disease (IBD).BACKGROUND

[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Inflammatory Bowel Disease (IBD), encompassing conditions such as Crohn's disease and ulcerative colitis, is characterized by chronic gut inflammation that causes symptoms like abdominal pain, frequent bowel movements, fatigue, and a significantly reduced quality of life. Patients diagnosed with moderate to severe IBD often face the challenge of managing symptoms with treatments that are only partially effective and frequently accompanied by adverse side effects. The existing therapies primarily target inflammation systemically, but they do not address the root causes of IBD, such as impaired mucosal healing and dysbiosis of the gut microbiota.

[0004] Inflammation in the gut leads to damage of the mucosal lining, impairing the gut's ability to heal and disrupts the balance of beneficial microbes, further exacerbating the disease. This damage can result in severe complications such as ulcerations, strictures, and long-term tissue destruction. In addition to causing discomfort, these complications increase the burden of the disease on the patient and healthcare system. There is an urgent need for therapies that can be directly applied to the affected areas of the gut, promote healing of the mucosal lining, reduce inflammation, and restore gut health.

[0005] Currently available treatments, including systemic immunosuppressants and biologies, are associated with significant side effects, limited effectiveness, and high costs. These treatments also fail to restore a healthy gut microbiota or provide long-term mucosal regeneration, which are essential for the management of IBD. Consequently, there is a growing need for targeted, localized, and natural therapeutic alternatives that can specifically address these issues, offering more effective and sustainable solutions for individuals suffering from IBD.

[0006] To address these challenges, a significant technology gap by offering an off- the-shelf, lyophilized formulation of probiotic-derived extracellular vesicles (EVs) encapsulated in a thermo-responsive polymer matrix. The targeted delivery and sustained release of therapeutic EVs directly at the site of inflammation provide localized treatment, reducing systemic side effects and offering a more efficient therapeutic approach.OBJECTS OF THE PRESENT DISCLOSURE

[0007] An object of the present disclosure is to provide a sprayable formulation for the enhanced treatment of Inflammatory Bowel Disease (IBD), utilizing probiotic-derived extracellular vesicles (EVs) encapsulated in a thermo-responsive polymer matrix.

[0008] Another object of the present disclosure is to incorporate probiotic-derived EVs from the commercially available VSL#3 probiotic blend, ensuring targeted therapeutic benefits for IBD treatment.

[0009] Another object of the present disclosure is to encapsulate the probiotic EVs in a thermo-responsive copolymer matrix made from poly (N-isopropyl acrylamide) and hyaluronic acid, enabling controlled release and targeted delivery at the site of inflammation.

[0010] Another object of the present disclosure is to offer the formulation as an off- the-shelf, lyophilized (freeze-dried) powder for easy storage, long shelf life, and convenience in rehydration and application.

[0011] Another object of the present disclosure is to provide a formulation that forms a gel-like coating over inflamed areas of the gut upon application, leveraging thermo- responsive behavior at physiological temperatures for localized treatment.

[0012] Another object of the present disclosure is to promote mucosal healing and reduce inflammation by combining the immune -modulatory and regenerative properties of probiotic EVs and the thermo-responsive copolymer matrix.SUMMARY

[0013] The present disclosure relates generally to the field of targeted gastrointestinal therapeutics and, more specifically, to a lyophilized, off-the-shelf formulation comprising probiotic-derived extracellular vesicles (EVs) encapsulated within a thermo-responsive Poly(N-isopropylacrylamide)-hyaluronic acid (PNIPAM-HA) copolymer matrix. The formulation is designed for the treatment of inflammatory bowel disease (IBD), including conditions such as Crohn’s disease and ulcerative colitis, by providing localized delivery ofanti-inflammatory and mucosal healing agents directly to inflamed regions of the gastrointestinal tract.

[0014] An aspect of the present disclosure relates to a therapeutic delivery system for the localized treatment of inflammatory bowel disease (IBD), including a lyophilized formulation configured for reconstitution before administration. The formulation includes a plurality of probiotic-derived extracellular vesicles (EVs) obtained from a probiotic blend including Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus delbrueckii, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, and Streptococcus thermophilus, encapsulated within a thermo-responsive copolymer matrix comprising Poly(N -isopropylacrylamide) (PNIPAM) having a lower critical solution temperature of approximately 32°C and hyaluronic acid (HA) configured to provide mucoadhesive properties, antioxidant activity, and tissue regenerative support.

[0015] Upon reconstitution and administration via non-invasive spray delivery or endoscopic application to an inflamed gastrointestinal site, the thermo-responsive matrix undergoes a sol-to-gel transition at body temperature, enabling localized retention and controlled, sustained release of the extracellular vesicles at the site of inflammation. The PNIPAM component exhibits a phase transition between 30°C and 37°C, providing thermo- responsive behavior, while the lyophilized formulation remains stable for up to six months at approximately 4°C without significant loss of protein content. The formulation further exhibits antioxidant properties to scavenge reactive oxygen species and reduce oxidative stress at the inflamed site, thereby minimizing systemic exposure and associated side effects, and providing enhanced therapeutic efficacy in the treatment of IBD, including Crohn’s disease and ulcerative colitis.

[0016] Another aspect of the present disclosure relates to a method for treating inflammatory bowel disease (IBD) in a subject in need thereof is provided, including rehydrating a lyophilized formulation that includes a plurality of probiotic-derived extracellular vesicles (EVs) obtained from a probiotic blend comprising Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus delbrueckii, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, and Streptococcus thermophilus, the EVs being encapsulated within a thermo-responsive copolymer matrix including Poly(N-isopropylacrylamide) (PNIPAM) and hyaluronic acid (HA).

[0017] In an embodiment, the method further includes administering the rehydrated formulation to an inflamed gastrointestinal site of the subject via non-invasive spray delivery or endoscopic administration, and allowing the thermo-responsive copolymer matrix toundergo a sol-to-gel transition at body temperature to provide localized retention and controlled release of the extracellular vesicles at the site of inflammation. In certain embodiments, the method further includes administering the formulation post-gastrointestinal surgery to promote mucosal regeneration and prevent infection, thereby enhancing therapeutic outcomes.

[0018] Various objects, features, aspects, and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures, in which like numerals represent similar components.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0020] FIG. 1 illustrates an exemplary representation of a therapeutic delivery system for localized treatment of inflammatory bowel disease (IBD), in accordance with an embodiment of the present disclosure.

[0021] FIG. 2 illustrates an exemplary representation for A. Fourier transform infrared (FTIR) spectroscopy of the monomer and synthesized polymers, B. nuclear magnetic resonance (NMR) spectroscopy of the synthesized polymers, C. Lower critical solution temperature (LCST) curve of the PNIPAM — HA copolymer showing its lower critical solution temperature, in accordance with an embodiment of the present disclosure.

[0022] FIG. 3 illustrates an exemplary representation for A. Anaerobic culture of the VSL#3 organisms and isolation of the EVs by ultrafdtration and ultracentrifugation, B. Scanning electron microscopy image of the freshly isolated EVs, C. Dynamic light scattering analysis of the freshly isolated EVs showing EV size of around lOOnm, D. Zeta potential analysis of the freshly isolated EVs which was found to be -20mV, in accordance with an embodiment of the present disclosure.

[0023] FIG. 4 illustrates an exemplary representation of A. Digital image of freshly prepared PNIPAM-HA copolymer and probiotic -derived EV formulation, B. Image of the lyophilized formulation, C. Scanning electron microscopy of the lyophilized formulation, D. Dynamic light scattering showing EV size around 55-60nm, E. Zeta potential analysis oflyophilized EVs, with a value of -20mV, in accordance with an embodiment of the present disclosure.

[0024] FIG. 5 illustrates an exemplary representation of A. Cell viability showing no significant difference between fresh and lyophilized PNIPAM-HA copolymer with probiotic EVs; B. DCF-DA assay showing similar antioxidant properties; C. DPPH assay showing comparable ROS scavenging; D.-F. Immunomodulatory analysis (iNOS, CD 163, IL-6) showing no significant difference between fresh and lyophilized formulations, in accordance with an embodiment of the present disclosure.

[0025] FIG. 6 illustrates an exemplary representation of A) Zeta potential of lyophilized-stored EVs ( — 18 mV) indicating stability; B) DLS analysis showing size ~50 nm; C) SEM of lyophilized-stored polymer+ProEV; D) Protein content comparison of fresh, lyophilized, and stored EVs showing no significant difference; E) Cell viability posttreatment with all three formulations showing no significant change; F) DPPH assay showing similar ROS scavenging efficiency across groups; G) DCF-DA assay showing reduced DCF fluorescence in fresh, lyophilized, and stored formulations, in accordance with an embodiment of the present disclosure.

[0026] FIG. 7 illustrates an exemplary flow diagram of a method for treating inflammatory bowel disease (IBD), in accordance with an embodiment of the present disclosure.DETAILED DESCRIPTION

[0027] The following is a detailed description of embodiments of the disclosure depicted in the accompanying drawings. The embodiments are in such detail as to clearly communicate the disclosure. If the specification states a component or feature “may”, “can”, “could”, or “might” be included or have a characteristic, that particular component or feature is not required to be included or have the characteristic.

[0028] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

[0029] While embodiments of the present disclosure have been illustrated and described, it will be clear that the invention is not limited to these embodiments only. Numerous modifications, changes, variations, substitutions, and equivalents will be apparentto those skilled in the art, without departing from the spirit and scope of the invention, as described in the claim.

[0030] The present disclosure is enabled by a process that allows preparation, storage, and application of a lyophilized therapeutic formulation comprising probiotic-derived extracellular vesicles (EVs) encapsulated within a thermo-responsive PNIPAM-HA copolymer matrix. The formulation is prepared by isolating EVs from a probiotic blend, incorporating them into a PNIPAM-HA matrix, and lyophilizing the mixture to obtain a stable powder. This lyophilized formulation retains its structural integrity and therapeutic activity for at least six months at 4°C. Before administration, the formulation is rehydrated to form a sprayable or injectable solution that undergoes sol-to-gel transition at body temperature, enabling localized retention, mucoadhesion, and controlled release of EVs at the site of gastrointestinal inflammation. The process ensures compatibility, stability, and ease of clinical application for treating inflammatory bowel disease.

[0031] FIG. 1 illustrates an exemplary representation of a therapeutic delivery system for localized treatment of inflammatory bowel disease (IBD), in accordance with an embodiment of the present disclosure.

[0032] In an embodiment, referring to FIG. 1, a therapeutic delivery system 100 (interchangeably referred to as a system 100, hereinafter) for localized treatment of inflammatory bowel disease (IBD) involves the development of a convenient, off-the-shelf lyophilized formulation 102 powder that contains a plurality of probiotic-derived extracellular vesicles (EVs) 104 encapsulated within a thermo-responsive copolymer matrix 106 composed of Poly-N-isopropyl acrylamide (PNIPAM) and hyaluronic acid (HA). This powder is designed to be easily stored, transported, and rehydrated when needed.

[0033] Furthermore, once the powder is rehydrated and applied to the colon via endoscopy, it undergoes a phase transition from a sol (liquid) state to a gel at body temperature. This phase change allows the lyophilized formulation 102 to form a physical gel-like coating over the inflamed tissue, providing a localized and sustained release of the encapsulated EVs 104 directly at the site of inflammation.

[0034] In an embodiment, the hyaluronic acid-based thermo-responsive matrix 106 enhances the adhesion of the formulation to the gut lining, promoting a gradual release of the EVs 104 at the inflamed area. This localized delivery ensures that the therapeutic agents (the EVs) remain at the site where they are most needed, while also augmenting the healing and regeneration of the mucosal tissue. The EVs 104, in combination with hyaluronic acid, workto reduce chronic inflammation and promote the repair of the gut lining, which is essential for maintaining the gut’s natural defense and function.

[0035] As an off-the-shelf lyophilized product, this formulation provides a highly flexible and convenient solution for both healthcare providers and patients. Its easy rehydration and application make it ideal for targeted drug delivery via endoscopy, offering a focused therapeutic effect directly at the site of inflammation, which minimizes systemic side effects. This localized treatment approach makes the formulation an efficient, patient- friendly, and effective solution for managing Inflammatory Bowel Disease (IBD).

[0036] In an exemplary embodiment, the present disclosure provides a lyophilized formulation 102 composed of probiotic-derived extracellular vesicles (EVs) 104 encapsulated in a thermo-responsive PNIPAM-HA polymer matrix 106, designed specifically for treating inflammatory bowel disease (IBD). The formulation, which is shelf-stable and off-the-shelf, can be easily rehydrated and applied via a non-invasive spray or endoscopically, delivering targeted therapy directly to the inflamed areas of the gut.

[0037] In an exemplary embodiment, the EVs 104, sourced from the VSL#3 probiotic blend, contain key therapeutic agents such as anti-inflammatory molecules, immune- modulating factors, and microbial signaling molecules. Upon application, these EVs 104 reduce local inflammation, promote mucosal healing, and restore gut microbiota balance, crucial in managing IBD. The PNIPAM-HA matrix is designed to undergo a phase transition from liquid to gel at body temperature (~32°C), enabling sustained and controlled release of EVs. This transition ensures that the therapeutic agents stay localized at the site of inflammation, enhancing their efficacy and minimizing systemic side effects.

[0038] Furthermore, hyaluronic acid (HA) in the matrix 106 provides mucoadhesive properties, ensuring the formulation adheres to the gut lining and prolongs residence time, which further enhances its therapeutic action. The lyophilized powder form of the product allows for easy storage, transport, and handling without the need for refrigeration, making it a convenient solution for clinical use. Upon rehydration, the formulation can be administered directly to inflamed areas of the gastrointestinal tract, either through a sprayable delivery system or an endoscopic method for more precise targeting.

[0039] In an exemplary embodiment, the lyophilized formulation 102 is particularly useful for patients with conditions like Crohn’s disease and ulcerative colitis, where inflammation and mucosal damage are prevalent. Beyond IBD, the technology can be expanded to treat other gastrointestinal disorders requiring localized drug delivery. It also has the potential to be applied in post-surgical recovery, where promoting mucosal regenerationand reducing inflammation is critical. The product’s versatility and patient-friendly design provide a novel approach to addressing the unmet needs in IBD treatment and gastrointestinal health.

[0040] FIG. 2 illustrates an exemplary representation for A. Fourier transform infrared (FTIR) spectroscopy of the monomer and synthesized polymers, B. nuclear magnetic resonance (NMR) spectroscopy of the synthesized polymers, C. Lower critical solution temperature (LCST) curve of the PNIPAM — HA copolymer showing its lower critical solution temperature, in accordance with an embodiment of the present disclosure.

[0041] Referring to FIG. 2, the experimental method 200 for synthesis and characterization of the copolymer formulation begins with the synthesis of amino-terminated poly (N -isopropylacrylamide) (PNIPAM) through radical polymerization. In the process, N- isopropylacrylamide (NIPAM) and N-tert-butylacrylamide (NTBAM) are dissolved in degassed water and polymerized in the presence of ammonium persulfate (APS) and cysteamine hydrochloride (AET HC1) as redox initiators, under a nitrogen atmosphere at 29°C with constant stirring for six hours.

[0042] Furthermore, once the reaction is complete, the mixture is dialyzed against deionized water to remove unreacted monomers and impurities. Next, the graft copolymer P(NIPAM-co-NTBAM)-HA is synthesized by coupling the amino-terminated copolymer with hyaluronic acid (HA) using EDC and NHS as coupling agents. HA is prepared by dialysis of its sodium salt form against 0.01 M HC1, followed by lyophilization. The coupling reaction occurs at a pH of ~5 for 24 hours at room temperature, and the resulting product is purified by dialysis to remove excess reagents and by-products. The copolymer’s structure and composition are confirmed using Fourier-transform infrared (FTIR) spectroscopy, where the copolymers (PNIPAM, P(NIPAM-co-NTBAM)-NH2, and P(NIPAM-co-NTBAM)-HA) are analyzed in the 400-4,000 cm range. Additionally, proton nuclear magnetic resonance (1H NMR) spectroscopy is performed with deuterated water as a solvent, and the spectra are analyzed using MNOVA software. The lower critical solution temperature (LCST) of the copolymer is measured by observing the absorbance of a 0. 1 wt% aqueous solution at 540 nm over a temperature range of 25-40°C using a Cary 60 UV-Vis spectrophotometer. This comprehensive synthesis and characterization method 100 can ensure the development of a thermo-responsive copolymer matrix with controlled properties for drug delivery applications.

[0043] FIG. 3 illustrates an exemplary representation for A. Anaerobic culture of the VSL#3 organisms and isolation of the EVs by ultrafiltration and ultracentrifugation, B.Scanning electron microscopy image of the freshly isolated EVs, C. Dynamic light scattering analysis of the freshly isolated EVs showing EV size of around lOOnm, D. Zeta potential analysis of the freshly isolated EVs which was found to be -20mV, in accordance with an embodiment of the present disclosure.

[0044] Referring to FIG. 3, the method 300 for the VSL#3 probiotics may be cultured for approximately 24 hours at 37°C in Lactobacillus MRS broth medium under anaerobic conditions. Once the optical density (OD) reached 1, bacterial cells and debris were removed by centrifugation at 9000 xg for 20 minutes. The supernatant is then filtered through 0.22 pm filters to remove any remaining larger particles. To enrich for extracellular vesicles (EVs), the filtered medium may be subjected to ultrafiltration using an Amicon® 100 kDa filter. The resulting concentrate underwent two hours of ultracentrifugation at 150,000xg to isolate the EVs. Following this, the precipitate may be centrifuged again for one hour at 150,000xg, after which the EVs were resuspended in sterile PBS and stored at -80°C for future use. The isolated EVs are characterized using various techniques, including dynamic light scattering (DLS) to measure particle size, zeta potential analysis to assess surface charge, and scanning electron microscopy (SEM) to examine the morphology of the vesicles. The method 200 may be allowed for a detailed characterization of the probiotic-derived EVs, which are key to the formulation in subsequent steps.

[0045] FIG. 4 illustrates an exemplary representation of A. Digital image of freshly prepared PNIPAM-HA copolymer and probiotic -derived EV formulation, B. Image of the lyophilized formulation, C. Scanning electron microscopy of the lyophilized formulation, D. Dynamic light scattering showing EV size around 55-60nm, E. Zeta potential analysis of lyophilized EVs, with a value of -20mV, in accordance with an embodiment of the present disclosure.

[0046] Referring to FIG. 4, experimental method 400, a 2.5% copolymer formulation can first prepared in phosphate-buffered saline (PBS). To this copolymer solution, 100 pg of extracellular vesicles (EVs) are added and gently mixed at 4°C to ensure uniform distribution. Trehalose, a cryoprotectant, is then incorporated into the formulation to a final concentration of 2% to protect the EVs during the lyophilization process. The mixture is poured into a Petri dish and refrigerated at -80°C for 2 hours before being lyophilized for 12 hours, resulting in a white, flaky matrix composed of the copolymer and probiotic EVs. After lyophilization, the matrix is rehydrated and diluted. The rehydrated formulation is then characterized using dynamic light scattering (DLS) to measure the particle size, zeta potential to assess surface charge, and scanning electron microscopy (SEM) to examine themorphology of the lyophilized copolymer-EV blend. The method 300 analyses provided insights into the properties and structure of the final formulation, essential for its intended use as a therapeutic agent.

[0047] FIG. 5 illustrates an exemplary representation of A. Cell viability showing no significant difference between fresh and lyophilized PNIPAM-HA copolymer with probiotic EVs; B. DCF-DA assay showing similar antioxidant properties; C. DPPH assay showing comparable ROS scavenging; D.-F. Immunomodulatory analysis (iNOS, CD 163, IL-6) showing no significant difference between fresh and lyophilized formulations, in accordance with an embodiment of the present disclosure.

[0048] Referring to FIG. 5, the method 500, the functionality of the off-the-shelf lyophilized formulation was compared to that of a freshly prepared formulation by assessing several key properties, including cell viability, proliferation, antioxidant potential, and immunomodulatory activity. Cell viability was evaluated using the MTT assay on HT-29 cells, a human colon cancer cell line, to determine the impact of the formulations on cell survival. The antioxidant potential was measured using two assays: DPPH (2,2-diphenyl-l- picrylhydrazyl) and DCF-DA (2',7'-dichlorofluorescein diacetate), which assess the ability of the formulations to scavenge free radicals and reduce oxidative stress in HT-29 cells.

[0049] Furthermore, to evaluate immunomodulatory potential, murine RAW 264.7 macrophages are first treated with lipopolysaccharide (LPS) to induce a proinflammatory Ml phenotype, and then exposed to both the off-the-shelf and freshly prepared formulations. Gene expression analysis may be performed on markers associated with Ml and M2 macrophage phenotypes to assess the formulations’ effects on immune modulation. These tests collectively provided insights into the therapeutic efficacy of the lyophilized formulation, especially in terms of its ability to modulate inflammation and promote healing in the context of inflammatory diseases.

[0050] FIG. 6 illustrates an exemplary representation of A) Zeta potential of lyophilized-stored EVs ( — 18 mV) indicating stability; B) DLS analysis showing size ~50 nm; C) SEM of lyophilized-stored polymer+ProEV; D) Protein content comparison of fresh, lyophilized, and stored EVs showing no significant difference; E) Cell viability posttreatment with all three formulations showing no significant change; F) DPPH assay showing similar ROS scavenging efficiency across groups; G) DCF-DA assay showing reduced DCF fluorescence in fresh, lyophilized, and stored formulations, in accordance with an embodiment of the present disclosure.

[0051] Referring to FIG. 6. the experimental method 600, comparative evaluation of fresh and six-month stored lyophilized probiotic vesicles encapsulated in a thermo-responsive copolymer matrix. In accordance with an embodiment of the present disclosure, a lyophilized formulation comprising a polymer and probiotic-derived extracellular vesicles (ProEV) may be stored at a temperature of approximately 4°C for a duration of six months. Upon completion of the storage period, the formulation was subjected to rehydration and subsequently evaluated for its physicochemical and biological properties.

[0052] In an embodiment, the rehydrated formulation is subjected to multiple dilutions to facilitate characterization through zeta potential analysis, dynamic light scattering (DLS), and scanning electron microscopy (SEM). Protein content within the formulation was quantified using a bicinchoninic acid (BCA) assay. Further, cytocompatibility of the formulation is assessed via an MTT assay employing an HT29 cell line. Antioxidant and reactive oxygen species (ROS) scavenging potential of the formulation was determined through a 2,2-diphenyl-l-picrylhydrazyl (DPPH) assay. Additionally, a dichlorofluorescein diacetate (DCF-DA) assay is performed using HT29 cells to confirm the antioxidant activity of the formulation.

[0053] FIG. 7 illustrates an exemplary flow diagram of a method for treating inflammatory bowel disease (IBD), in accordance with an embodiment of the present disclosure.

[0054] In an embodiment, referring to FIG. 7, the method 700 for treating inflammatory bowel disease (IBD), where the method 700 involves multiple steps; at step 702, the method 700 involves rehydrating a lyophilized formulation 102) prior to administration, where the formulation includes a plurality of probiotic -derived extracellular vesicles (EVs) 104 obtained from a probiotic blend including Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus delbrueckii, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, and Streptococcus thermophilus. The EVs 104 are encapsulated within a thermo-responsive copolymer matrix 106 including Poly(N-isopropylacrylamide) (PNIPAM), which exhibits a lower critical solution temperature of about 32°C, and hyaluronic acid (HA), which provides mucoadhesive properties, antioxidant activity, and tissue regenerative support. Upon rehydration, the formulation transitions into a liquid or gel form suitable for administration, enabling controlled and localized release of the encapsulated extracellular vesicles at the target site.

[0055] Continuing further, at step 704, the method 700 involves administering the rehydrated formulation to an inflamed gastrointestinal site of a subject using either a non-invasive spray delivery system or an endoscopic administration route. In one implementation, the rehydrated formulation, prepared from the lyophilized composition containing probiotic- derived extracellular vesicles encapsulated in a thermo-responsive PNIPAM-HA copolymer matrix 106, is delivered as a fine mist or spray directly onto the inflamed mucosal surface through a non-invasive applicator. Alternatively, for deeper or hard-to-reach lesions, the formulation is introduced via an endoscopic device to ensure precise placement at the site of inflammation. Upon administration, the thermo-responsive matrix undergoes sol-to-gel transition at body temperature, forming a localized gel layer that adheres to the mucosal tissue and enables sustained, controlled release of extracellular vesicles for reducing inflammation, modulating immune response, and promoting mucosal healing.

[0056] Continuing further, at step 706, the method 700 involves allowing the thermo- responsive copolymer matrix (106) to undergo a sol-to-gel transition upon exposure to body temperature after administration to the gastrointestinal site. The matrix 106, including Poly(N-isopropylacrylamide) (PNIPAM) and hyaluronic acid (HA), remains in a liquid state during rehydration and delivery but transitions into a gel-like state at approximately 32°C to 37°C, corresponding to physiological conditions. This phase transition enables localized retention of the formulation at the site of inflammation, preventing premature clearance and ensuring prolonged contact with the mucosal surface. The gelled matrix facilitates controlled and sustained release of the encapsulated probiotic-derived extracellular vesicles (EVs) 104 over an extended period, allowing continuous delivery of anti-inflammatory and regenerative agents for effective treatment of inflammatory bowel disease.

[0057] In summary, the present disclosure introduces off-the-shelf lyophilized formulation containing probiotic-derived extracellular vesicles (EVs) from the VSL#3 probiotic blend, encapsulated in a thermo-responsive PNIPAM-HA copolymer matrix. The formulation is designed for targeted therapy in inflammatory bowel disease (IBD), offering a shelf-stable product that can be easily rehydrated for use. The thermo-responsive matrix reacts to body temperature, transitioning into a gel that adheres to the gut lining, allowing for localized and sustained release of the EVs at the site of inflammation. The EVs provide antiinflammatory effects and promote mucosal healing, while the hyaluronic acid enhances muco-adhesion and tissue repair. The formulation can be applied via spray or endoscopically, ensuring precise delivery and minimizing systemic side effects. The invention’s innovative aspect lies in the integration of lyophilized probiotic EVs with a thermo-responsive copolymer matrix, offering a controlled and effective treatment for IBD that is distinct from current therapies.

[0058] While the foregoing describes various embodiments of the present disclosure, other and further embodiments of the present disclosure may be devised without departing from the basic scope thereof. The scope of the present disclosure is determined by the claims that follow. The present disclosure is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the present disclosure when combined with information and knowledge available to the person having ordinary skill in the art.ADVANTAGES OF THE PRESENT DISCLOSURE

[0059] The present disclosure ensures long-term stability without the need for refrigeration, making it easy to store, transport, and handle in clinical settings.

[0060] The present disclosure ensures that the EVs are released directly at the site of inflammation in the gut, minimizing systemic exposure and side effects.

[0061] The present disclosure promotes strong adhesion to the gut lining, facilitating prolonged residence time and enhancing the therapeutic effect at the site of inflammation.

[0062] The present disclosure a focused treatment for inflammatory bowel disease (IBD), particularly for conditions like Crohn’s disease and ulcerative colitis, by delivering anti-inflammatory and mucosal healing agents directly to the inflamed regions.

[0063] The present disclosure can be applied as a spray or delivered via endoscopic methods, offering flexibility in treatment options, particularly for hard-to-reach areas in the gastrointestinal tract.

Claims

We Claim:

1. A therapeutic delivery system (100) for localized treatment of inflammatory bowel disease (IBD), wherein the system (100) comprises: a lyophilized formulation (102) configured for reconstitution before administration, the lyophilized formulation (102) comprising: a plurality of probiotic-derived extracellular vesicles (EVs) (104) obtained from a probiotic blend comprising Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus delbrueckii, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, and Streptococcus thermophilus; and a thermo-responsive copolymer matrix (106) encapsulating the plurality of probiotic-derived extracellular vesicles (EVs) (104), wherein the thermo-responsive copolymer matrix (106) comprises:Poly(N-isopropylacrylamide) (PNIPAM) exhibiting a lower critical solution temperature (LCST) of about 32°C; and hyaluronic acid (HA) adapted to provide mucoadhesive properties, antioxidant activity, and tissue regenerative support.

2. The system (100) as claimed in claim 1, wherein the system (100) is configured for non-invasive spray delivery or endoscopic administration for localized therapeutic effect in the treatment of inflammatory bowel diseases, comprising Crohn’s disease and ulcerative colitis.

3. The system (100) as claimed in claim 1, wherein upon rehydration and application to an inflamed gastrointestinal site, the thermo-responsive matrix undergoes a sol-to-gel transition at body temperature to provide localized retention of the extracellular vesicles, enabling controlled and sustained release of the vesicles at the site of inflammation.

4. The system (100) as claimed in claim 1, wherein the PNIPAM undergoes phase transition from liquid to gel state at a temperature between 30°C and 37°C, enabling thermo- responsive behavior at body temperature.

5. The system (100) as claimed in claim 1, wherein the lyophilized formulation (102) is stable for a period of up to six months at a temperature of about 4°C without significant loss of protein content.

6. The system (100) as claimed in claim 1, wherein the lyophilized formulation (102) further comprises antioxidant properties to scavenge reactive oxygen species (ROS) and reduce oxidative stress at the site of inflammation.

7. The system (100) as claimed in claim 1, wherein the system (100) is configured for localized release of extracellular vesicles, thereby reducing systemic exposure and associated side effects.

8. A method (700) for treating inflammatory bowel disease (IBD) in a subject in need thereof, wherein the method (700) comprises the steps of: rehydrating (702) a lyophilized formulation (102) comprises: a plurality of probiotic-derived extracellular vesicles (EVs) (104) obtained from a probiotic blend comprising Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus delbrueckii, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, and Streptococcus thermophilus; and a thermo-responsive copolymer matrix (106) encapsulating the extracellular vesicles (104), wherein the matrix comprises Poly(N- isopropylacrylamide) (PNIPAM) and hyaluronic acid (HA); administering (704) the rehydrated formulation to an inflamed gastrointestinal site of the subject through a non-invasive spray delivery or endoscopic administration; and allowing (706) the thermo-responsive copolymer matrix (106) to undergo a sol-to-gel transition at body temperature for localized retention and controlled release of the extracellular vesicles.

9. The method (700) as claimed in claim 8, further comprises administering the formulation post-gastrointestinal surgery for mucosal regeneration and infection prevention.

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