A novel targeted delivery system for treating severe ulcerative colitis and use thereof

Abstract

The application belongs to the field of biological medicine, and discloses a novel targeted delivery system for treating severe ulcerative colitis and application, which comprises the following steps: S101, intracellular gelation process: the Akk bacteria are amplified to the logarithmic growth phase, and after centrifugal resuspension, the bacteria are incubated with BPQDs-NH2 and gel buffer; the components are promoted into cells through a single freeze-thaw cycle; S102, cross-linking reaction: a cross-linking reaction is initiated by using 365nm, 1600mJ / cm² ultraviolet light, and a targeted delivery system GAkk@BP system is obtained through purification. The GAkk@BP oral delivery system constructed in the application has high ROS scavenging capacity, can significantly reduce the intestinal epithelial cell damage induced by ROS, and effectively protect the cell function; in addition, the intracellular gelation modification has realized the precise regulation of Akk proliferation, successfully avoided the risk of excessive colonization, and laid a solid foundation for the subsequent optimization of the intracellular gelation probiotic delivery system, the in-depth analysis of the proliferation blocking / metabolic isolation and the mechanism of the synergistic treatment of UC.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a novel targeted delivery system and its application for treating severe ulcerative colitis. Background Technology

[0002] Severe ulcerative colitis (UC) is a chronic inflammatory disease affecting the colonic mucosa and submucosa. In severe cases, it can lead to persistent diarrhea, bloody stools, abdominal pain, fever, anemia, weight loss, and even life-threatening complications such as intestinal perforation and toxic megacolon. Currently, commonly used traditional drugs for treating UC, such as 5-aminosalicylic acid (5-ASA), hormones, and immunosuppressants, can alleviate symptoms but cannot stop mucosal inflammation or disease progression. Furthermore, severe short-term (systemic immunosuppression and the risk of opportunistic infections) and long-term (Cushing's syndrome, diabetes, and osteoporosis) adverse reactions limit the application of most traditional drugs.

[0003] Akkermansia muciniphila (Akk) is a Gram-negative anaerobic bacterium belonging to the phylum Verrucomicrobia. It is not only found in the human gut but has also been developed as a probiotic supplement. Clinical studies have shown that oral administration of inactivated Akk bacteria or the Akk bacteria outer membrane protein Amuc_1100 can regulate CD8... + Cytotoxic T cells (CTLs) improve colitis and prevent related colorectal cancers with good safety profiles. However, orally administered Akk bacteria proliferate before reaching the intestinal mucus layer, increasing the risk of disordered proliferation and bacterial translocation infection in the damaged intestinal microenvironment.

[0004] Black phosphorus quantum dots are novel nanomaterials with good drug loading capacity, strong photothermal effects, and excellent biocompatibility, making them ideal as drug carriers. However, black phosphorus quantum dots exhibit poor stability under physiological conditions, are prone to degradation and aggregation, and require additional coating or modification for further application. Furthermore, black phosphorus quantum dots lack active targeting ability to target organs in vivo. Imparting active targeting ability through surface modification and other strategies can effectively improve their drug delivery efficacy.

[0005] Therefore, this invention proposes a novel targeted delivery system and its application for treating severe ulcerative colitis. Summary of the Invention

[0006] This invention aims to provide a novel targeted delivery system and application for treating severe ulcerative colitis. AKK bacteria are loaded onto aminated black phosphorus quantum dots, and then polyethylene glycol diacrylate (PEG-DA) is used to form a polymeric hydrogel network physical barrier within the AKK bacterial cytoplasm through in-situ free radical polymerization. This barrier blocks the proliferation of AKK bacteria, while the aminated black phosphorus quantum dots remove excess ROS and increase the metabolic rate of Akk. Both work synergistically to drive the repair of the damaged intestinal mucosal barrier, thus addressing the technical problem of the lack of effective treatment options for severe ulcerative colitis.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a novel targeted delivery system for treating severe ulcerative colitis, comprising the following steps:

[0009] S101, Intracellular Gelization Process

[0010] Akk bacteria were amplified to the logarithmic growth phase, centrifuged and resuspended, and then mixed with BPQDs-NH2 and gel buffer. After incubation of the mixture, a single freeze-thaw cycle was used to promote the entry of the components into the cells.

[0011] The gel buffer is composed of PEG-DA at a concentration of 0.7~0.9 mL / mL and a photoinitiator at a concentration of 550~650 mg / mL;

[0012] S102, cross-linking reaction

[0013] The targeted delivery system GAkk@BP was obtained by initiating a cross-linking reaction and purification using 365nm, 1600mJ / cm² ultraviolet light.

[0014] Preferably, in step S101, the OD of the Akk bacteria during the logarithmic growth phase... 600 The molecular weight is 0.6~0.8, the average molecular weight of the PEG-DA is 700, and the photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone.

[0015] Preferably, the viable concentration of Akk bacteria in the mixture is 1×10⁻⁶. 6 The concentration of CFU / mL and BPQDs-NH2 is 15~25ug / mL.

[0016] Preferably, the incubation is performed at 35±2℃ with constant rotation at 0.125Hz for at least 12 hours, and the crosslinking reaction takes 3~5 minutes.

[0017] Preferably, the single freeze-thaw cycle consists of pre-cooling with supercooled methanol at a temperature not higher than -80°C for at least 2 minutes, freezing at a temperature not higher than -80°C for at least 10 minutes, and then thawing at a temperature not lower than 30°C for at least 5 minutes; the purification is performed by killing ungelled Akk bacteria with 350~450μg / mL of carbenicillin.

[0018] Preferably, the preparation method of BPQDs-NH2 includes the following steps:

[0019] Preparation of S201 and BPQDs

[0020] Add NMP to BP powder at a ratio of 35-45 mg: 10 mL, grind in a glove box for at least 30 min; transfer to 50 mL centrifuge tubes, add 50 mL of NMP so that each tube contains about 35-45 mg of BP powder, sonicate in an ice bath for at least 6 h; after gradient centrifugation, wash several times to obtain BPQDs.

[0021] Preparation of S202 and BPQDs-NH2

[0022] Polyethyleneimine was electrostatically adsorbed with BPQDs at room temperature for at least 6 hours, purified by dialysis, and lyophilized to obtain BPQDs-NH2.

[0023] Preferably, the ultrasound cycle is 7 seconds with a 5-second interval, the gradient centrifugation is 6000-8000 rpm for 4-8 minutes, the supernatant is poured out, and centrifugation is continued at 11000-13000 rpm for 25-40 minutes.

[0024] Preferably, the volume-to-mass ratio of the polyethyleneimine to BPQDs is 80-120:1, and the molecular weight of the polyethyleneimine is 70,000-90,000 da.

[0025] Secondly, the present invention provides a novel targeted delivery system prepared using the above-described preparation method.

[0026] Thirdly, the present invention provides the application of a novel targeted delivery system in the preparation of drugs for treating severe ulcerative colitis.

[0027] Compared with existing technologies, the beneficial effects of this solution are:

[0028] 1. The GAkk@BP oral delivery system constructed in this invention exhibits excellent biocompatibility at the cellular level and has no obvious cytotoxicity; at the same time, it has a highly efficient ROS scavenging ability, which can significantly reduce ROS-induced intestinal epithelial cell damage and effectively protect cell function; in addition, intracellular gelation modification has achieved precise regulation of Akk proliferation, successfully avoiding the risk of over-colonization.

[0029] 2. The core mechanisms of action of the GAkk@BP oral delivery system constructed in this invention include: 1) Physical barrier and safety control: The molecular crowding effect and physical confinement effect generated by the intracellular gel network can physically block the division and proliferation of Akk bacteria, thereby eliminating the risk of disordered proliferation and bacterial translocation infection in the damaged intestinal microenvironment; at the same time, it constructs a spatial steric barrier to block the contact of mobile genetic elements (plasmids) between probiotics and conditionally pathogenic bacteria, and cuts off the horizontal transfer (HGT) of drug resistance genes (ARGs). 2) Metabolic retention and synergistic therapy: The unique semi-permeable network of the hydrogel allows for the intake of nutrients and the continuous secretion of functional short-chain fatty acids (SCFAs). After local decomposition and release, BPQDs clear excess ROS and block oxidative stress, and enhance the metabolic rate of Akk through electron transport; the two synergistically drive the repair of the damaged intestinal mucosal barrier and the reconstruction of local immune homeostasis (Treg / Th17 balance). Attached Figure Description

[0030] Figure 1 Morphological characterization of the targeted delivery system GAkk@BP (A is the evaluation of in-situ photocrosslinking gelation characteristics (a is gel buffer; b is gel buffer, c is gel buffer mixed with 2×FTG medium; d is crosslinking gel buffer, e is crosslinking gel buffer mixed with 2×FTG medium); B, C, D, and E are TEM images of BPQDs, BPQDs-NH2, Akk, and GAkk@BP, respectively; F and G are TEM images of Akk and GAkk after decellularization with 1% SDS, respectively; H and I are the potentiograms and particle size distributions of BPQDs, BPQDs-NH2, Akk, GAkk, and GAkk@BP, respectively).

[0031] Figure 2 The intracellular hydrogel network of GAkk@BP (A is a confocal microscope image; B is a laser confocal scan of GAkk@BP (morphology of GAkk@BP (top); 3D laser confocal scan of GAkk@BP (middle); magnified 3D laser confocal scan of GAkk@BP (bottom)).

[0032] Figure 3 The results of the validation of the proliferation inhibition characteristics of GAkk@BP are shown in Figure A (images of Akk and GAkk@BP after 72 h of plate culture, and growth curves of Akk, GAkk and GAkk@BP after 72 h of culture in FTG liquid medium (n=3)).

[0033] Figure 4The results of GAkk@BP's in vitro antioxidant activity are shown in Figure 1 (A shows the effect of different concentrations of BPQDs on the survival rate of cells in the oxidative stress injury model; B shows the effect of BPQDs, GAkk, and GAkk@BP on the survival rate of cells in the oxidative stress injury model; C shows the expression of ROS in RAW264.7 cells after treatment with BPQDs, GAkk, GAkk+BP, and GAkk@BP by DCFH-DA staining; and D shows the cell nuclei labeled with DAPI staining).

[0034] Figure 5 The results of GAkk@BP in vitro anti-inflammatory treatment are shown in Figure 1 (A shows the effect of different concentrations of BPQDs on the expression levels of anti-inflammatory factors in inflammatory model cells; B shows the effect of BPQDs, GAkk, and GAkk@BP on the expression levels of anti-inflammatory factors in inflammatory model cells; C shows CD206 immunostaining on M2 macrophages; and D shows iNOS immunostaining on M1 macrophages).

[0035] Figure 6 Results of GAkk@BP in vitro intestinal mucosal repair;

[0036] Figure 7 Effects of GAkk@BP on body weight and other parameters in DSS-induced acute colitis mice (A: representative photographs of rectal bleeding in each group; B: changes in body weight in each group; C: disease activity index scores in each group).

[0037] Figure 8 Effects of GAkk@BP on colon and spleen in DSS-induced acute colitis mice (A: effect on colon tissue length; B: effect on spleen index; C: digital images of cecum-colon tissue and spleen; D and E: representative histological images of colon tissue stained with H&E and AB-PAS after treatment with different formulations, scale bar: 200 μm; *P < 0.05, **P < 0.01, ***P < 0.001).

[0038] Figure 9 Effects of GAkk@BP on serum inflammatory factor levels in mice with DSS-induced acute colitis;

[0039] Figure 10 To demonstrate the alpha diversity of the fecal microbiome using the Shannon and Chaos indices after GAkk@BP treatment;

[0040] Figure 11 To demonstrate the β diversity of the fecal microbiome after GAkk@BP treatment using NMDS and PCoA plots;

[0041] Figure 12Relative abundance at the family level for Muribaculaceae, Lachnospiraceae, and Enterobacteriaceae Shigella;

[0042] Figure 13 To analyze the phylum-level composition of the gut microbiota in each group of mice;

[0043] Figure 14 Distribution of the top 10 bacteria by family abundance in the gut microbiota of each group of mice;

[0044] Figure 15 The level of SCFAs in each group of mice (n=3);

[0045] Figure 16 The results of the in vitro safety assessment of GAkk@BP are as follows: (A shows the effect of different concentrations of BPQDs, GAkk, and GAkk@BP on the viability of HT29 and NCM460 cells; B shows the staining of live / dead cells after co-incubation of different concentrations of GAkk@BP with RAW264.7 and NCM460 cells, respectively).

[0046] Figure 17 The results of the in vivo safety assessment of GAkk@BP are shown in Figures A and B (hematological analysis of mice in each group), and Figure C (H&E staining of important organs of mice in each group, n=3, scale bar: 200μm). Detailed Implementation

[0047] To facilitate understanding of the present invention by those skilled in the art, the technical solution of the present invention will be further described in detail below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0048] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0049] The present invention discloses a novel targeted delivery system for treating severe ulcerative colitis, comprising the following steps:

[0050] Step 1: Amination modification of BPQDs

[0051] Step 1.1, Preparation of BPQDs

[0052] Weigh out bulk black scaly BP crystals at a ratio of 35-45 mg: 10 mL and place them in a small mortar. Add NMP and grind in a glove box for at least 30 min. Transfer the powder to 50 mL centrifuge tubes and add 50 mL of NMP to each tube, so that each tube contains about 35-45 mg of BP powder. Sonicate in an ice bath for at least 6 h (7 s working time followed by 5 s intervals). Centrifuge at 6000-8000 rpm for 4-8 min, gently pour off the supernatant, and continue centrifuging at 11000-13000 rpm for 25-40 min. Wash with deoxygenated water 3-5 times to obtain BPQDs.

[0053] Step 1.2, Preparation of BPQDs-NH2

[0054] Polyethyleneimine (pH 7.4) with a molecular weight of 70,000 to 90,000 da was electrostatically adsorbed with BPQDs at room temperature for at least 6 hours at a volume-to-mass ratio of 80 to 120:1. After dialysis purification, the positively charged aminated BPQDs (BPQDs-NH2) were obtained by freeze-drying.

[0055] Step 2: Construction of the Targeted Delivery System GAkk@BP

[0056] Step 2.1, Intracellular Gelization Process

[0057] Akk bacteria were amplified to the logarithmic growth phase (OD) in an anaerobic workstation. 600 =0.6-0.8), after centrifugation and resuspending, according to 1×10 6 Incubate the sample at a ratio of 15–25 μg / mL with BPQDs-NH2 and gel buffer (containing 0.7–0.9 mL / mL PEG-DA and 550–650 mg / mL photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone) at 37°C with constant rotation for at least 12 h. Promote cell entry using a single freeze-thaw cycle (pre-cooling with supercooled methanol at no higher than -80°C for at least 2 min, freezing at no higher than -80°C for at least 10 min, and then thawing at no lower than 30°C for at least 5 min).

[0058] Step 2.2, Crosslinking reaction

[0059] Subsequently, a cross-linking reaction was initiated with 365 nm, 1600 mJ / cm² ultraviolet light for 3-5 min to induce in-situ free radical polymerization. After killing the ungelled Akk with carbenicillin (350-450 μg / mL), the targeted delivery system GAkk@BP was obtained.

[0060] The aforementioned targeted delivery system GAkk@BP includes the cornerstone of treatment (Akk bacteria) that utilizes the natural chemotactic colonization properties and electrostatic adsorption of the intestinal lesion mucus layer to achieve targeted delivery; functional nanomaterials (BPQDs-NH2, with a particle size of 2-5 nm) with excellent catalase-like and superoxide dismutase activities that can efficiently remove pathological reactive oxygen species (ROS); and an intracellular three-dimensional gel network of polymer hydrogel network formed by in-situ free radical polymerization of polyethylene glycol diacrylate (PEG-DA) as a crosslinking monomer inside the bacterial cytoplasm.

[0061] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific application examples described herein are merely illustrative and not intended to limit the invention. However, it should not be construed that the scope of the invention is limited to the following embodiments. Various substitutions and modifications made based on ordinary technical knowledge and common practices in the art without departing from the above-described technical concept of this invention should be included within the scope of protection of this invention.

[0062] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0063] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0064] Example 1: Construction of a Targeted Delivery System

[0065] 1. Experimental materials and reagents

[0066] AKK bacteria were purchased from Beijing Beina Chuanglian Biotechnology Research Institute.

[0067] Preparation of gel buffer: Dissolve 36 mg of fluorescein O,O'-diacrylate in 1 mL of DMSO to obtain a fluorescein solution with a concentration of 36 mg / mL;

[0068] Dissolve 0.75g of hydroxy-4′-(2-hydroxyethoxy)-2-methylpropenone (photoinitiator) in DMSO and vortex for 2 minutes to obtain a photoinitiator solution with a concentration of 750mg / mL;

[0069] Mix 800 µl of PEG-DA (Mn: 700) with 160 µl of photoinitiator solution, and homogenize by vortexing. Add 28 µl of fluorescein solution to the homogenized mixture, and add DMSO to bring the total volume to 1 mL, so that the final concentration of fluorescein is approximately 1 mg / mL, the final concentration of photoinitiator is 600 mg / mL, and the final concentration of PEG-DA is 0.8 mL / mL.

[0070] The energy of UVP crosslinking agent is 1600 mJ / cm. 2 Time: 5 min. For efficient and precise crosslinking of hydrogel matrices. Before the experiment, the UVP crosslinking agent needs to be preheated for one cycle at the same energy level.

[0071] 2. Construction of a novel targeted delivery system

[0072] 2.1 Preparation and Amination Modification of BPQDs

[0073] 2.1.1 Preparation of BPQDs

[0074] An ultrasonic-assisted liquid-phase exfoliation method was used. NaOH was added to ultra-dry N-methylpyrrolidone (NMP) and ultrasonically alkalized the solvent. Combined with a solvothermal reaction, the mixture was ultrasonically treated in an ice bath for 6-8 hours, followed by gradient centrifugation purification to prepare BPQDs with uniform particle size (2-5 nm). Specifically, the process included:

[0075] Weigh 40 mg of bulk black scaly crystals (BP) into a small mortar, add 10 mL of NMP, and grind in a glove box for 30 min. Transfer the mixture to a 50 mL centrifuge tube and add 50 mL of NMP. Then sonicate in an ultrasonicator on ice for 6 h (7 s cycle, 5 s interval). After sonication, centrifuge at 7000 rpm for 5 min, then gently discard the supernatant. Continue centrifuging at 12000 rpm for 30 min, and wash three times with deoxygenated water to obtain BPQDs.

[0076] 2.1.2 Amination-modified BPQDs

[0077] Subsequently, 1% polyethyleneimine (PEI, pH 7.4, 80000 da) was electrostatically adsorbed with BPQDs at room temperature for 6 hours at a volume-to-mass ratio of 100:1. After dialysis purification, positively charged aminated BPQDs (BPQDs-NH2) were obtained and lyophilized for later use to enhance their cell membrane penetration.

[0078] 2.2 Intracellular gelation process and system construction

[0079] Akk bacteria were amplified to the logarithmic growth phase (OD) in an anaerobic workstation. 600 =0.6-0.8), after centrifugation and resuspending, it was mixed with BPQDs-NH2 and gel buffer. The viable concentration of Akk bacteria in the mixture was 1×10⁻⁶. 6The concentration of CFU / mL and BPQDs-NH2 was 20 μg / mL. The mixture was incubated at 37°C with constant rotation at 0.125 Hz for 12 h. A single freeze-thaw cycle (pre-cooling with supercooled methanol at -80°C for 2 min, freezing at -80°C for 10 min, and thawing at 30°C for 5 min) was used to promote cell entry. Subsequently, a cross-linking reaction was initiated for 5 min with 1600 mJ / cm² UV light (light source: UVP cross-linking CL-3000L - Longwave (365 nm), 115 V) to trigger in-situ free radical polymerization. After killing ungelled Akk with carbenicillin (400 μg / mL), the purified product was obtained, which is the targeted delivery system GAkk@BP.

[0080] Example 2 Preparation of GAkk

[0081] Akk bacteria were amplified in an anaerobic workstation to achieve an OD value of 0.8-1.0. Cells were harvested by centrifugation at 4000g for 10 minutes at 20°C, resuspended in culture medium to a cell density of 0.2g / mL, and transferred to 1.5mL centrifuge tubes. The cells were mixed with gel buffer and incubated at a constant 0.125Hz at 37°C for 30 minutes, rotating on a centrifuge shaft with the tube inverted each time. After incubation with the hydrogel buffer, the centrifuge tubes were immersed in supercooled methanol and rapidly frozen at -80°C for 2 minutes. They were then incubated at -80°C for 10 minutes, thawed in a 30°C bath, and immediately centrifuged at 6800g for 10 minutes at 20°C. The cells were then washed twice with fresh culture medium. The centrifuge tubes were then heated with 1600 mJ / cm³. 2 Ultraviolet light was used to crosslink bacteria (light source: UVP crosslinking CL-3000L-Longwave (365 nm), 115V). After UV irradiation, cells were centrifuged at 6800g for 10 min at 20°C and washed twice with 1×PBS buffer. After final washing and centrifugation, cells were resuspended and incubated in 2YTP medium (37°C, constant rotation at 0.125Hz on the axis of rotation), and an appropriate antibiotic was added, carbicillin (400 μg / mL) to kill actively dividing non-hydrogel bacteria. After incubation, cells were harvested (6800g, 10 min, 20°C), washed with 1×PBS, and resuspended in medium to obtain GAkk.

[0082] Example 3 Characterization of the Targeted Delivery System GAkk@BP

[0083] This embodiment evaluates the in-situ photocrosslinking gelation characteristics of the gel buffer from Example 1 and performs morphological verification and key physical property characterization on the targeted delivery system GAkk@BP constructed in Example 1.

[0084] 1. Evaluation of in-situ photocrosslinking gelation characteristics

[0085] Fluid thioglycollate medium (FTG) and gel buffer, alone or in mixture, were placed under ultraviolet light (365 nm, 1600 mJ / cm²) to initiate cross-linking. Visual images confirmed that the gel buffer successfully underwent a phase transition after photocross-linking, indicating that this gel buffer possesses excellent photocross-linking and in-situ gelation capabilities. Figure 1 (A in the middle).

[0086] 2. Morphological characterization

[0087] The particle size and potential of BPQDs, BPQDs-NH2, Akk, and GAkk@BP in Example 1 and GAkk in Example 2 were analyzed. TEM images of BPQDs, BPQDs-NH2, Akk, and GAkk@BP were analyzed. After Akk and GAkk outer membranes were peeled off using 1% SDS, the intracellular polymeric gel network structure of Akk bacteria was precisely resolved using high-resolution transmission electron microscopy (HR-TEM).

[0088] Table 1. Particle size and potential of BPQDs, BPQDs-NH2, Akk, GAkk, and GAkk@BP

[0089]

[0090] Dynamic light scattering results showed that after amination modification, the surface Zeta potential of BPQDs changed from -29.22±4.01 mV to 22.97±2.46 mV, with no significant change in particle size; subsequently, after loading BPQDs and undergoing intracellular gelation, the overall particle size of GAkk@BP increased (Table 1). Figure 1 The H and I in the text are mentioned. Transmission electron microscopy (TEM) observation shows that GAkk@BP retains the original outline of Akk, but the presence of a gel core within the cell increases electron transmittance, resulting in higher light transmittance. Figure 1 The B, C, D, and E in the data preliminarily confirm the successful construction of the GAkk@BP delivery system.

[0091] Example 3: Functional Verification of the Targeted Delivery System GAkk@BP

[0092] 1. Confirmation of the GAkk@BP intracellular hydrogel network

[0093] To visually verify the formation of hydrogels inside bacteria, a fluorescent monomer (O,O'-diacrylate) was introduced to label the gel buffer. Laser confocal microscopy and 3D scanning image analysis were performed on Akk, Akk incubated with gel buffer but not UV crosslinked, and GAkk@BP (UV crosslinked for 3 min and 5 min). The results showed that green fluorescence was uniformly distributed inside GAkk cells. Figure 2 (A and B in the text). Furthermore, after peeling the bacterial membrane with 1% SDS, TEM observations showed that natural Akk exhibited significant swelling and rupture, while GAkk retained an intact and insoluble internal gel skeleton (…). Figure 1 The F and G values ​​in the data confirm that gelation occurs inside the bacterial cell.

[0094] 2. Verification of the proliferation-blocking properties of GAkk@BP

[0095] The combined plate spread method was used to observe the culture status of Akk and GAkk@BP on ready-to-use potato agar plates for 72 consecutive hours, and to measure the OD values ​​of Akk, GAkk, and GAkk@BP in FTG liquid medium at different time points within 72 hours. 600 , and plotted the growth curve for 72 consecutive hours.

[0096] By comparing the growth curves of different systems in the culture medium, it was found that the growth of GAkk was significantly inhibited (see...). Figure 3 This indicates that the molecular crowding effect and physical confinement generated by the intracellular in situ gel network block bacterial division and reduce the risk of opportunistic infections caused by over-colonization and translocation of live bacteria.

[0097] Example 4: In vitro antioxidant, repair-promoting, and anti-inflammatory effects of GAkk@BP

[0098] 1. Evaluation of targeted uptake and antioxidant activity

[0099] A H2O2-induced oxidative stress injury model of RAW264.7 cells was constructed. Cells treated with physiological saline served as the positive control group (H2O2 group), while untreated RAW264.7 cells treated with physiological saline served as the negative control group (Control group). The effects of different concentrations of BPQDs on cell viability were compared. Furthermore, the effects of the same concentrations of BPQDs, GAkk@BP, and GAkk on cell viability were also compared. The ability of internalized GAkk@BP to scavenge intracellular ROS was assessed using the DCFH-DA probe, and the intracellular spatial localization of BPQDs was confirmed using DAPI double staining.

[0100] The results showed that GAkk significantly enhanced the targeted uptake of BPQDs by intestinal epithelial cells. Figure 4 Based on this, GAkk@BP can not only efficiently remove excess ROS induced by H2O2 (D). Figure 4 The C in it also has a protective effect against H2O2 damage in HT29 cells. Figure 4 (A and B in the text).

[0101] 2. Evaluation of GAkk@BP's in vitro anti-inflammatory effects

[0102] A lipopolysaccharide (LPS)-induced inflammation model of RAW264.7 macrophages was constructed. LPS-induced inflammation model cells treated with physiological saline served as the positive control group (denoted as the LPS group), while untreated RAW264.7 macrophages treated with physiological saline served as the negative control group (denoted as the Control group). The effects of different concentrations of BPQDs treated with LPS-induced inflammation model cells (denoted as BPQDs-2.5, BPQDs-5.0, BPQDs-10.0, and BPQDs-20.0) on the expression levels of intracellular anti-inflammatory factors in each group were compared. Furthermore, the effects of BPQDs, GAkk, GAkk+BP, and GAkk@BP treated with the same concentrations on the expression levels of intracellular anti-inflammatory factors in LPS-induced model cells (denoted as the BPQDs group, GAkk group, GAkk+BP group, and GAkk@BP group, respectively) on the expression levels of intracellular anti-inflammatory factors in each group were compared. CD206 staining and iNOS staining were performed on each group of cells to confirm the inflammation type of the macrophages.

[0103] Macrophage polarization regulation, anti-inflammatory effects, and mucosal repair: An inflammation model was constructed by stimulating RAW264.7 macrophages with LPS. The results showed that GAkk@BP could effectively inhibit macrophage polarization towards pro-inflammatory M1 and conversion towards anti-inflammatory M2. Figure 5 (C and D in the text). Simultaneously, its inhibitory effect on inflammatory factors was significantly better than that of free BPQDs and single bacteria, suggesting that the intracellular gelation system may enhance the stability of BPQDs and synergistically reduce inflammation. Figure 5 (A and B in the text).

[0104] 3. Evaluation of GAkk@BP in vitro repair promotion

[0105] A lipopolysaccharide (LPS)-induced ulcerative colitis model was constructed in NCM460 cells. LPS-induced ulcerative colitis model cells treated with physiological saline served as the positive control group (denoted as LPS group), while untreated NCM460 cells treated with physiological saline served as the negative control group (denoted as Control group). The effects of BPQDs, GAkk, GAkk+BP, and GAkk@BP treatments on cell repair in LPS-induced model cells (denoted as BPQDs group, GAkk group, GAkk+BP group, and GAkk@BP group, respectively) were compared using a cell scratch assay.

[0106] The results showed that GAkk@BP could promote the migration of LPS-damaged NCM460 cells to a certain extent, demonstrating a good ability to promote intestinal mucosal healing (see...). Figure 6 ).

[0107] Example 5: In vivo efficacy evaluation of GAkk@BP

[0108] 1. Animal model construction

[0109] A severe UC model simulating clinical severe mucosal loss and antibiotic selection pressure was established by using 6-8 week old C57BL / 6 female mice and pretreating them for 7 days with high concentration (4%) DSS in free drinking water combined with ampicillin / streptomycin dual antibiotics.

[0110] 2. In vivo pharmacodynamic evaluation

[0111] Severe ulcerative colitis (UC) model mice were randomly divided into 6 groups. One group of severe UC model mice and healthy mice were administered PBS buffer by gavage for 7 consecutive days, serving as the blank control group (Control) and the DSS model group (DSS). Another group of severe UC model mice were administered 5-ASA positive drug by gavage for 7 days (5-ASA). The other four groups of severe UC model mice were treated with Akk 1×10 6 Mice were administered CFU / mL doses of Akk, GAkk, a physical mixture of GAkk and BP, and GAkk@BP via gavage for 7 days. Mice in each group were euthanized on day 14. Body weight and stool hematoma were dynamically recorded for DAI scoring. Colon length and spleen index were measured at the endpoint via anatomical dissection. Colonic tissue was stained with H&E and AB-PAS to assess crypt structure and inflammatory infiltration.

[0112] The results showed that the DSS group mice experienced significant weight loss throughout the experiment, accompanied by rectal bleeding. Figure 7 In case A), significant weight loss, elevated DAI score, shortened colon length, and changes in spleen index were observed. Figure 7 B, C and Figure 8(A and B in the text). In contrast, mice in both the Akk and GAkk groups showed some degree of symptom relief, weight gain, and controlled bleeding; however, the GAkk@BP group showed the most significant reduction in DAI score, marked recovery in colon length, and pathological experimental results further confirmed the efficacy of GAkk@BP. Figure 8 The levels of inflammatory factors (C, D, and E) were significantly improved (see [reference needed]). Figure 9 Comparative studies revealed that Akk, GAkk, and GAkk@BP all had a certain degree of therapeutic effect on UC, with the GAkk@BP group showing the most significant improvement, fully demonstrating its significant therapeutic potential for UC.

[0113] 3. The effect of GAkk@BP on gut microbiota regulation in DSS-induced acute UC

[0114] To evaluate the regulatory effect of GAkk@BP on the gut microbiota, this study used 16S rRNA sequencing and targeted metabolomics to detect the composition of gut microbiota and the levels of SCFAs (acetic acid, propionic acid, butyric acid, etc.) in each group of mice.

[0115] The results showed that DSS treatment significantly reduced the Shannon and Chaos indices, indicating a decrease in gut microbiota richness and evenness; while after GAkk@BP intervention, both indices significantly recovered and approached those of the control group, indicating that it can improve gut microbiota diversity in UC mice (Figure 10). NMDS and PCoA results further indicated that GAkk@BP can promote a gut microbiota structure closer to that of the healthy control group (…). Figure 11 Regarding gut microbiota composition, GAkk@BP significantly increased the relative abundance of beneficial bacteria such as Muribaculaceae and Lachnospiraceae, and decreased the levels of potentially pathogenic bacteria such as Enterobacteriaceae and Shigella, demonstrating a good regulatory effect on the gut microbiota (see...). Figure 12 At the genus level, a similar trend was observed, with a significant increase in beneficial bacteria such as *Bifidobacterium* and *Muribaculaceae*, while a significant decrease in potentially pathogenic bacteria (see [link to article]). Figure 13 , 14 Furthermore, the levels of SCFAs such as butyrate, isobutyrate, and acetic acid were significantly decreased in the DSS group mice, while these metabolites were significantly restored after GAkk@BP treatment, reaching or even exceeding the levels of the control group. Figure 15 The results showed that GAkk@BP can effectively improve gut microbiota imbalance and promote the production of SCFAs, thus playing a positive therapeutic role.

[0116] Example 6: Biocompatibility of GAkk@BP

[0117] 1. In vitro safety study

[0118] This embodiment investigates the cytotoxicity of BPQDs, GAkk, and GAkk@BP by exploring the effects of different concentrations of BPQDs, GAkk, and GAkk@BP on the viability of HT29 and NCM460 cells, and also examines the live / dead cell staining after co-incubation of RAW264.7 and NCM460 cells with different concentrations of GAkk@BP.

[0119] The results showed that GAkk@BP could thrive at BPQDs concentrations of 0-20 μg / mL and GAkk viable cell concentrations of 1×10⁻⁶. 6 Good safety profile within the CFU / mL range (see [link]). Figure 16 ).

[0120] 2. In vivo safety assessment

[0121] Two groups of healthy mice were set up: a control group (denoted as Control) and an experimental group (denoted as GAkk@BP). The mice were administered PBS buffer and GAkk@BP (at an Akk1×10⁻⁶) by gavage, respectively. 6 The drug was administered via gavage at a dose of CFU / mL for 28 consecutive days. Complete blood cell counts and liver and kidney function biochemical indicators were measured in each group, and H&E staining of major organs was performed to confirm the safety of clinical translation.

[0122] After 28 consecutive days of treatment with GAkk@BP, healthy mice maintained normal levels of all blood and related biochemical indicators; H&E staining and pathological analysis of major organs showed no significant organic lesions (see [link to relevant documentation]). Figure 17 ).

[0123] In summary, the GAkk@BP oral delivery system constructed in this invention, through characterization and functional validation, demonstrates excellent biocompatibility at the cellular level and exhibits no significant cytotoxicity. It also possesses highly efficient ROS scavenging capabilities, significantly mitigating ROS-induced intestinal epithelial cell damage and effectively protecting cell function. Furthermore, intracellular gelation modification has achieved precise regulation of Akk proliferation, successfully avoiding the risk of over-colonization. These experimental results not only validate the feasibility of the design of intracellular gelled probiotics loaded with BPQDs but also confirm the protective delivery and synergistic therapy concept of this invention, laying a solid foundation for subsequent optimization of intracellular gelled probiotic delivery systems and in-depth analysis of the mechanisms of proliferation inhibition / metabolic isolation and synergistic therapy for UC.

[0124] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A novel targeted delivery system for treating severe ulcerative colitis, characterized in that, Includes the following steps: S101, Intracellular Gelization Process Akk bacteria were amplified to the logarithmic growth phase, centrifuged and resuspended, and then mixed with BPQDs-NH2 and gel buffer. After incubation of the mixture, a single freeze-thaw cycle was used to promote the entry of the components into the cells. The gel buffer contains PEG-DA at a concentration of 0.7~0.9 mL / mL and a photoinitiator at a concentration of 550~650 mg / mL; S102, cross-linking reaction The targeted delivery system GAkk@BP was obtained by initiating a cross-linking reaction and purification using 365nm, 1600mJ / cm² ultraviolet light.

2. The novel targeted delivery system for treating severe ulcerative colitis and its application according to claim 1, characterized in that, In step S101, the OD of the Akk bacteria during the logarithmic growth phase... 600 The molecular weight is 0.6~0.8, and the average molecular weight of the PEG-DA is 700; the photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone.

3. The novel targeted delivery system for treating severe ulcerative colitis according to claim 1, characterized in that, The viable concentration of Akk bacteria in the mixture was 1×10⁻⁶. 6 The concentration of CFU / mL and BPQDs-NH2 is 15~25 μg / mL.

4. A novel targeted delivery system for treating severe ulcerative colitis according to claim 1, characterized in that, The incubation is carried out at 35±2℃ with constant rotation at 0.125Hz for at least 12 hours, and the crosslinking reaction takes 3~5 minutes.

5. A novel targeted delivery system for treating severe ulcerative colitis according to claim 1, characterized in that, The single freeze-thaw cycle consists of pre-cooling with supercooled methanol at a temperature not higher than -80°C for at least 2 minutes, freezing at a temperature not higher than -80°C for at least 10 minutes, and then thawing at a temperature not lower than 30°C for at least 5 minutes; the purification process involves using 350~450 μg / mL of carbenicillin to kill ungelled Akk bacteria.

6. A novel targeted delivery system for treating severe ulcerative colitis according to claim 1, characterized in that, The preparation method of the BPQDs-NH2 includes the following steps. Preparation of S201 and BPQDs Add NMP to BP powder at a ratio of 35-45 mg: 10 mL, grind in a glove box for at least 30 min; transfer to 50 mL centrifuge tubes, add 50 mL of NMP so that each tube contains about 35-45 mg of BP powder, sonicate in an ice bath for at least 6 h; after gradient centrifugation, wash several times to obtain BPQDs. Preparation of S202 and BPQDs-NH2 Polyethyleneimine was electrostatically adsorbed with BPQDs at room temperature for at least 6 hours, purified by dialysis, and lyophilized to obtain BPQDs-NH2.

7. A novel targeted delivery system for treating severe ulcerative colitis according to claim 1, characterized in that, The ultrasound cycle is 7 seconds with a 5-second interval. The gradient centrifugation is performed at 6000-8000 rpm for 4-8 minutes, the supernatant is poured out, and centrifugation is continued at 11000-13000 rpm for 25-40 minutes.

8. A novel targeted delivery system for treating severe ulcerative colitis according to claim 1, characterized in that, The volume-to-mass ratio of the polyethyleneimine to BPQDs is 80-120:1, and the molecular weight of the polyethyleneimine is 70,000-90,000 da.

9. A novel targeted delivery system prepared by the preparation method according to any one of claims 1-8.

10. The use of the novel targeted delivery system of claim 9 in the preparation of a drug for treating severe ulcerative colitis.

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