A colon-targeted diagnosis and treatment integrated microcapsule material and a preparation method and application thereof

Abstract

The application discloses a colon-targeted diagnosis and treatment integrated microcapsule material and a preparation method and application thereof, and comprises the following steps: (1) mixing and reacting oppositely charged modified cellulose and a photosensitizer to obtain an electrostatically interacted cellulose-photosensitizer compound; (2) heating and gelatinizing oxidized starch, mixing the oxidized starch with a gelatin solution, adding the cellulose-photosensitizer compound and a polyphenol aqueous solution, fully stirring, and standing to obtain a preliminarily shaped microcapsule; and (3) placing the preliminarily shaped microcapsule in a refrigeration condition, fully aging and shaping, and then vacuum freeze-drying to obtain the colon-targeted diagnosis and treatment integrated microcapsule material. The oxidized starch-gelatin-polyphenol double crosslinking network is used as a wall material, and the cellulose-indocyanine green compound is loaded; the wall material remains stable in a stomach and small intestine environment, and specifically degrades and releases polyphenol and the photosensitizer compound at the front end of the colon, so that the functions of fluorescence / photoacoustic imaging and anti-inflammatory repair diagnosis and treatment integration are realized.

Description

Technical Field

[0001] This invention belongs to the field of colon diagnostic and therapeutic materials, specifically relating to a colon-targeted diagnostic and therapeutic integrated microcapsule material, its preparation method, and its application. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory bowel disease. Its lesions primarily affect the colonic mucosa and submucosa, exhibiting a continuous and diffuse distribution. It has become a major digestive system disease posing a serious threat to human health worldwide. The pathogenesis of UC is complex, involving the interaction of multiple factors such as genetic susceptibility, environmental factors, gut microbiota dysbiosis, and immune disorders. Its core pathological features include impaired intestinal mucosal barrier, activation of the chronic inflammatory cascade, and dysfunction of intestinal epithelial cells. Clinically, it mainly manifests as persistent or recurrent diarrhea, mucus and bloody stools, abdominal pain, and tenesmus, severely impacting patients' quality of life. Furthermore, long-term inflammatory stimulation can significantly increase the risk of colorectal cancer.

[0003] Currently, the clinical treatment of ulcerative colitis (UC) still faces many challenges. Traditional treatments such as aminosalicylic acids, glucocorticoids, and immunosuppressants, while providing some symptom relief, have limitations including slow onset of action, significant side effects with long-term use, easy development of drug resistance, and difficulty in achieving targeted intervention of the lesion. The advent of biologics and small-molecule targeted drugs has provided new options for UC treatment, but their high treatment costs, potential infection risks, and non-response issues in some patients limit their widespread application. Meanwhile, the accurate diagnosis and efficacy evaluation of UC also face bottlenecks: traditional colonoscopy, while the gold standard for diagnosis, is invasive, has poor patient compliance, and is difficult to perform real-time dynamic monitoring of intestinal inflammation; serological and fecal biomarker tests lack specificity and cannot accurately reflect the extent and severity of the lesion. Therefore, developing novel integrated diagnostic and therapeutic strategies that combine precise colonic imaging with targeted therapy has become a key direction urgently needing breakthroughs in the field of UC research.

[0004] Quaternized ammonium modified cellulose (QMCC), as a novel bio-based functional material, introduces cationic groups through quaternization modification. This not only retains cellulose's good biocompatibility, biodegradability, and mechanical properties but also endows it with excellent charge-electric interaction capabilities and structural stability, making it an ideal carrier platform for loading therapeutically active molecules. Indocyanine green (ICG), an FDA-approved near-infrared fluorescent dye, possesses advantages such as high fluorescence quantum yield, strong tissue penetration, and good biosafety, showing great potential in areas such as colon inflammation imaging and tumor detection. However, free ICG suffers from poor in vivo stability, rapid clearance, lack of tissue targeting, and susceptibility to degradation in the gastrointestinal environment, severely limiting its application in colon diagnosis and treatment. Summary of the Invention

[0005] To address the aforementioned issues, this invention constructs a colon-targeted therapeutic microcapsule material. This system uses QMCC as a carrier to load ICG, achieving efficient loading through the electrostatic interaction and physical encapsulation of quaternized groups with ICG. Simultaneously, a starch-gelatin complex cross-linked with EGCG is coated onto its surface. Utilizing the anti-acid properties of starch, the isoelectric point transition behavior of gelatin, and the antioxidant and anti-inflammatory activities of EGCG, a triple barrier is constructed, combining structural stability, targeted delivery, and synergistic therapeutic functions. This ensures the stability of the formulation within the gastric and small intestinal environments, guaranteeing precise delivery to the colonic lesion site.

[0006] The present invention adopts the following technical solution:

[0007] A method for preparing a colon-targeted diagnostic and therapeutic integrated microcapsule material includes the following steps:

[0008] (1) Modified cellulose with opposite charges is mixed and reacted with photosensitizer to obtain cellulose-photosensitizer complex with electrostatic interaction;

[0009] (2) Heat the oxidized starch to gelatinize it and mix it with the gelatin solution. Then add the cellulose-photosensitizer complex and polyphenol aqueous solution from (1), stir thoroughly, and finally let it stand to obtain the microcapsules that have been preliminarily shaped.

[0010] (3) The microcapsules that have been preliminarily shaped are placed under refrigeration conditions for sufficient aging and shaping, and then vacuum freeze-dried to obtain the integrated microcapsule material for colon-targeted diagnosis and treatment.

[0011] Preferably, the concentration of the gelatin solution in step (2) is 5% to 15% w / v; the mass ratio of oxidized starch to gelatin is 1:100 to 100:1. The gelatin is type A gelatin or type B gelatin.

[0012] Preferably, the oxidized starch in step (2) is carboxylated starch with a carboxyl content of 0.01% to 0.5%; and the polyphenol content is 0.1% to 20% w / v of the dry starch mass.

[0013] Preferably, the cellulose-photosensitizer complex in step (2) accounts for 0.1% to 10% of the total dry weight of starch.

[0014] Preferably, the modified cellulose in step (1) is a positively charged cellulose obtained by quaternary ammonium salt cationization modification; the photosensitizer is a negatively charged photosensitizer; and the mass ratio of the photosensitizer to the modified cellulose is 1:100 to 1:1.

[0015] Preferably, the cellulose in step (1) is cellulose derived from wood pulp or cotton pulp, with an average particle size of 20 nm to 200 μm, more preferably 20 to 100 μm; the photosensitizer is indocyanine green.

[0016] Preferably, the polyphenols in the polyphenol aqueous solution in step (2) are selected from at least one of tannic acid, epigallocatechin gallate, proanthocyanidins, gallic acid, and tea polyphenols; the polyphenol content is 0.1% to 20% w / v of starch dry basis mass.

[0017] Preferably, the oxidized starch in step (2) is oxidized starch obtained by oxidizing starch raw materials; the starch raw materials are selected from at least one of corn starch, potato starch, cassava starch and other starches; the oxidizing agent for the oxidation treatment is at least one of sodium periodate, sodium hypochlorite and hydrogen peroxide, to obtain carboxylated starch with a carboxyl content of 0.01% to 0.5%.

[0018] Preferably, the mixing reaction in step (1) is carried out under light-protected conditions, with stirring for 4 to 12 hours at pH 6 to 10 and temperature 0 to 55°C, and the concentration of metal salt in the reaction system is 0 to 20 mg / L.

[0019] Preferably, the heating and gelatinization conditions in step (2) are as follows: the oxidized starch is dispersed in water and stirred and gelatinized at 80-95°C for 20-60 minutes, and then cooled to room temperature.

[0020] Preferably, the refrigeration conditions in step (3) are: temperature 2℃~8℃, refrigeration time 12~48 hours; the vacuum freeze-drying conditions are: temperature -50℃~-80℃, vacuum degree less than 10 Pa, drying time 24~72 hours.

[0021] Preferably, the pH value of the mixed reaction in step (1) is 7-9, the temperature is 15-35℃, and the sodium chloride concentration is 0-10 mg / L; the preparation of the modified cellulose is as follows: cellulose is swollen in an alkali metal hydroxide solution, and then 2,3-epoxypropyltrimethylammonium chloride is added as a quaternizing agent. The reaction is carried out at 20-80℃ for 1-5 hours, and after neutralization, washing and drying, the modified cellulose is obtained.

[0022] The method described yields a colon-targeted therapeutic microcapsule material comprising a wall material composed of an oxidized starch-gelatin-polyphenol crosslinked network, and a cellulose-photosensitizer complex dispersed within the wall material. The microcapsule material specifically degrades in the anterior colon, releasing polyphenols and a microcrystalline cellulose-indocyanine green complex.

[0023] The application of the colon-targeted diagnostic and therapeutic integrated microcapsule material in the preparation of products for the diagnosis and treatment of colonic diseases (such as colitis).

[0024] Compared with the prior art, the present invention has the following outstanding advantages:

[0025] (1) The present invention loads a negatively charged photosensitizer onto positively charged modified microcrystalline cellulose through electrostatic interaction, which effectively improves the stability of the photosensitizer and solves the defects of free photosensitizer being easily degraded and having a short half-life in the gastrointestinal environment.

[0026] (2) The present invention uses oxidized starch, gelatin and polyphenols to construct a double cross-linked network wall material. The carbonyl group of oxidized starch and the amino group of gelatin form Schiff bases and electrostatic cross-links. The polyphenols form hydrogen bonds and hydrophobic interactions with oxidized starch and gelatin, thus constructing a double cross-linked network in the microcapsule wall. The double cross-linked network endows the microcapsules with excellent mechanical strength and structural integrity, making them stable in the strong acid and enzymatic environment of gastric juice and intestinal juice, realizing the on-demand release of active substances in the anterior colon and degradation in colonic juice.

[0027] (3) The polyphenol components selected in this invention (such as epigallocatechin gallate, etc.) have the dual functions of crosslinking agent and therapeutic agent. On the one hand, they participate in the construction of the wall material crosslinking network and enhance the structural stability of microcapsules; on the other hand, after the microcapsules degrade, they are released to the lesion site in the colon to exert their own anti-inflammatory, antioxidant and intestinal mucosal protective effects, and work synergistically with the photoacoustic / fluorescence imaging function of indocyanine green to realize the integrated diagnosis and treatment of colonic diseases.

[0028] (4) The microcapsule material preparation method of the present invention is simple and efficient. All raw materials used are natural or FDA-approved biodegradable materials with good biocompatibility, low cost, and easy to scale up production. It has broad application prospects in imaging diagnosis and targeted therapy of colon diseases. Attached Figure Description

[0029] Figure 1 The figures show the in vitro digestion release curves of the materials prepared in Examples 1-5; the left figure shows the EGCG release rate, and the right figure shows the QMCC-ICG release rate.

[0030] Figure 2 The images shown are scanning electron microscope (SEM) images of different embodiments and comparative examples; the top image shows different magnifications of 6%EGCG-MC, and the bottom image shows different magnifications of 6%EGCG-MC@QMCC-ICG.

[0031] Figure 3 The DAI score of mice after intervention is shown.

[0032] Figure 4 This is a graph showing the content of lipoprotein (NGAL) in mouse feces after intervention.

[0033] Figure 5 In vivo fluorescence imaging images of mice after intervention in different embodiments and comparative examples.

[0034] Figure 6 Photoacoustic endoscopic images of mice after intervention in different embodiments and comparative examples.

[0035] Figure 7 Photoacoustic micrographs of mice after intervention in different embodiments and comparative examples.

[0036] Figure 8 These are sections of organ tissues (heart, liver, spleen, lung, and kidney) from mice after intervention. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0038] Microcrystalline cellulose was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and was of analytical grade.

[0039] 2,3-Epoxypropyltrimethylammonium chloride was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and was of analytical grade.

[0040] Indocyanine green, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., analytical grade.

[0041] Epigallocatechin gallate (EGCG) was purchased from Shanghai Maclean Biotechnology Co., Ltd., and was of analytical grade.

[0042] Type A gelatin was purchased from Shanghai McLean Biochemical Technology Co., Ltd., and was of analytical grade.

[0043] Preparation of oxidized starch: 10 g of corn starch was weighed and added to water to prepare a 45% (w / w) suspension. The mixture was stirred at 300 rpm to maintain uniform mixing, and the pH was adjusted to 8.0. Then, a sodium hypochlorite solution with an effective chlorine content of 9% (w / w, based on dry starch) was added dropwise, and the reaction was continued at room temperature for 2 hours. After the reaction, the resulting precipitate was repeatedly washed with deionized water until no precipitation occurred upon addition of AgNO3 solution to the supernatant. The precipitate was then washed once with anhydrous ethanol. The final product was dried, ground, and passed through an 80-mesh sieve to obtain the oxidized starch sample.

[0044] Example 1

[0045] Step 1: 5 g of microcrystalline cellulose and 0.25 g of sodium hydroxide were thoroughly mixed in a mortar at room temperature for 5 min, then cooled to room temperature. The mixture was then transferred to a 100 mL beaker and 5 mL of water and 30 mL of DMSO were added. The mixture was sonicated for 10 min, followed by the addition of 10 mL of 2,3-epoxypropyltrimethylammonium chloride solution (200% effective content based on dry microcrystalline cellulose). The dispersion was sonicated for 30 min. The beaker was then heated in a 65°C water bath with mechanical stirring at a rate of 500 r / min for 4 h. After the reaction was complete, 45 mL of 95% ethanol was added to terminate the reaction. The mixture was washed four times with ethanol to remove any unreacted reagents and byproducts. Finally, it was dried in a 60°C oven to obtain sample QMCC-1. Pipette 30 mL of 400 mg / L QMCC stock solution and 40 mL of 200 mg / L ICG stock solution into a 250 mL Erlenmeyer flask. Add deionized water to maintain a total solution volume of 100 mL. Stir the solution at a constant speed of 125 rpm for 8 h under the conditions of NaCl salt content of 10 mg / L, temperature of 45℃ and pH of 8. Continue to centrifuge to remove impurities and freeze-dry to obtain microcrystalline cellulose-indocyanine green QMCC@ICG.

[0046] Step 2: Weigh 2 g of oxidized corn starch and add deionized water to prepare a 10% starch solution (w / w). Heat and stir at 80°C until dissolved. Weigh 2 g of gelatin A and add deionized water to prepare a 10% gelatin solution (w / w). Heat and stir at 35°C until dissolved. Mix the starch solution and gelatin solution at a 3:2 ratio and stir for 5 min to form a relatively homogeneous pre-microcapsule mixture. Add QMCC-ICG (1% wt based on dry starch) and continue stirring for 20 min. Add EGCG powder (6% wt based on dry starch) and stir for 2 h. Let the preliminarily formed microcapsule precursor stand for 2 h to allow it to complete preliminary structural solidification and network assembly at room temperature.

[0047] Step 3: The preliminarily shaped microcapsule samples were transferred to a refrigerated environment at 4°C and stored for 12 hours for sufficient aging and shaping. Then, they were frozen at -80°C for 24 hours and then freeze-dried in a vacuum freeze dryer to obtain the colon-targeted diagnostic and therapeutic integrated material 6%EGCG-MC@QMCC-ICG.

[0048] Example 2

[0049] The difference between this embodiment and embodiment 1 is that the EGCG content in step 2 is 2%wt (based on dry starch), and the colon-targeted diagnostic and therapeutic integrated material 2%EGCG-MC@QMCC-ICG is prepared.

[0050] Example 3

[0051] The difference between this embodiment and embodiment 1 is that the EGCG content in step 2 is 4%wt (based on dry starch), and the colon-targeted diagnostic and therapeutic integrated material 4%EGCG-MC@QMCC-ICG is prepared.

[0052] Example 4

[0053] The difference between this embodiment and embodiment 1 is that the EGCG content in step 2 is 8%wt (based on dry starch), and the colon-targeted diagnostic and therapeutic integrated material 8%EGCG-MC@QMCC-ICG is prepared.

[0054] Example 5

[0055] The difference between this embodiment and embodiment 1 is that the EGCG content in step 2 is 10%wt (based on dry starch), and the colon-targeted diagnostic and therapeutic integrated material 10%EGCG-MC@QMCC-ICG is prepared.

[0056] Example 6

[0057] The difference between this embodiment and Embodiment 1 is that step 1 involves preparing the colon-targeted diagnostic and therapeutic integrated material 6%EGCG-MC@QMCC1-ICG using 10 mL of 2,3-epoxypropyltrimethylammonium chloride solution (with an effective content of 100% based on microcrystalline cellulose dry basis).

[0058] Example 7

[0059] The difference between this embodiment and Embodiment 1 is that step 1 involves preparing the colon-targeted diagnostic and therapeutic integrated material 6%EGCG-MC@QMCC3-ICG using 10 mL of 2,3-epoxypropyltrimethylammonium chloride solution (with an effective content of 300% based on microcrystalline cellulose dry basis).

[0060] Example 8

[0061] The difference between this embodiment and Embodiment 1 is that the ratio of oxidized starch to gelatin in step 2 is 1:4, thus obtaining the colon-targeted diagnostic and therapeutic integrated material 6%EGCG-MC1@QMCC-ICG.

[0062] Example 9

[0063] The difference between this embodiment and embodiment 1 is that the ratio of oxidized starch to gelatin in step 2 is 2:3, thus obtaining the colon-targeted diagnostic and therapeutic integrated material 6%EGCG-MC2@QMCC-ICG.

[0064] Example 10

[0065] The difference between this embodiment and embodiment 1 is that the ratio of oxidized starch to gelatin in step 2 is 4:1, which yields the colon-targeted diagnostic and therapeutic integrated material 6%EGCG-MC3@QMCC-ICG.

[0066] Example 11

[0067] The difference between this embodiment and Embodiment 1 is that in step 2, polyphenols are replaced with anthocyanins to obtain the colon-targeted diagnostic and therapeutic integrated material 6%AN-MC@QMCC-ICG.

[0068] Example 12

[0069] The difference between this embodiment and Embodiment 1 is that indocyanine green is replaced with fluorescein in step 1 to prepare the colon-targeted diagnostic and therapeutic integrated material 6%EGCG-MC@QMCC-FLU.

[0070] Comparative Example 1

[0071] The difference between this comparative example and Example 1 is that no ICG was added in step 1 of this comparative example, and the sample of Comparative Example 1 was prepared with 6% EGCG-MC@QMCC.

[0072] Comparative Example 2

[0073] The difference between this comparative example and Example 1 is that this comparative example does not include the ICG loading preparation process in step 1, and in step 2, the ICG solution is directly added to obtain the 6% EGCG-MC@ICG sample of Comparative Example 2.

[0074] Comparative Example 3

[0075] The difference between this comparative example and Example 1 is that this comparative example did not undergo the preparation of microcapsules in step 2, thus obtaining the sample QMCC-ICG of Comparative Example 3.

[0076] Comparative Example 4

[0077] The difference between this comparative example and Example 1 is that no EGCG was added in step 2 of this comparative example, thus obtaining the sample of Comparative Example 4, 0%EGCG-MC@QMCC-ICG.

[0078] Comparative Example 5

[0079] The difference between this comparative example and Example 1 is that the oxidized corn starch in step 2 is replaced with ordinary corn starch, thus obtaining the sample 6%EGCG-MC4@QMCC-ICG of Comparative Example 5.

[0080] Comparative Example 6

[0081] The difference between this comparative example and Example 1 is that in step 2 of this comparative example, the microcapsules do not contain oxidized starch but only gelatin, resulting in sample 6% EGCG-GE@QMCC-ICG for Comparative Example 6.

[0082] Comparative Example 7

[0083] The difference between this comparative example and Example 1 is that: in step 2 of this comparative example, no gelatin is added to the microcapsules, and the 6% EGCG-OMS@QMCC-ICG sample of Comparative Example 7 is obtained.

[0084] This invention evaluates the structure, performance, and integrated diagnostic and therapeutic efficacy of the colon-targeted microcapsule for ulcerative colitis in the above embodiments and comparative examples, as detailed below:

[0085] 1. In vitro simulated digestion test method:

[0086] The specific procedures for simulating the in vitro digestion process are as follows: Accurately weigh 1.00 g (dry basis) of microcapsule material and add it to 20 mL of acetate-sodium acetate buffer. In the oral cavity simulation stage, salivary α-amylase with an enzyme activity of 7.5 U / mL was added to the system, and the pH was adjusted to 7.0 to simulate oral conditions. The system was then placed in a 37 ℃ constant-temperature shaker and shaken at 140 r / min for 2 min. In the gastric simulation stage, the pH was adjusted to 1.2, and pepsin was added. The system was continuously reacted in a 37 ℃ constant-temperature water bath shaker at 140 r / min for 2 h to simulate the gastric digestive environment. Subsequently, in the small intestine simulation stage, the pH was adjusted to 6.8, 5 mL of mixed enzyme solution was added, and the reaction continued for 6 h under the same shaking conditions to simulate the small intestine digestion process. Samples of 0.5 mL were taken at 2 min of oral digestion, 120 min of gastric digestion, and 240 min, 360 min, and 480 min of small intestine digestion, and immediately transferred to 20 mL of 70% ethanol solution for enzyme inactivation. Finally, the ethanol mixture was centrifuged at 5000 r / min for 5 min, and the supernatant was collected for testing.

[0087] (1) In vitro simulated EGCG release test

[0088] Standard curve preparation: Weigh 10 mg of EGCG standard, dissolve it in ethanol, and bring the volume to 100 ml to obtain a standard solution with a concentration of 0.1 mg / mL. Accurately pipette 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 ml into 10 ml amber volumetric flasks, add 1 ml of Folin-Ciocalteau reagent, mix well, add 2 ml of Na2CO3 solution (15%, w / v), mix thoroughly, and bring the volume to a final depth. Let stand for 1 h. Use distilled water without standard solution as a blank control. Measure the absorbance at 760 nm. Perform three parallel measurements for each sample. Plot the standard curve to obtain the regression linear equation.

[0089] Sample determination: Take 0.5 ml of the sample to be tested into a 10 ml volumetric flask, add 1 ml of Folin-Ciocalteau reagent, mix well, add 2 ml of Na2CO3 solution (15%, w / v), mix thoroughly, and make up to volume. Let stand for 1 h, using distilled water as a blank control, and measure the absorbance at 760 nm. Each sample is measured in triplicate. Substitute the values ​​into the standard curve to calculate the concentration C of EGCG. t The release rate of EGCG in different samples was calculated using the following formula:

[0090]

[0091] In the formula: C t : The concentration of the test sample;

[0092] V t : Total volume of the test sample (mL);

[0093] D: Dilution factor;

[0094] W t Sample mass (g)

[0095] A: EGCG content;

[0096] (2) The method for testing the release of QMCC-ICG in vitro is similar to that in (1).

[0097] 3. SEM scanning test

[0098] The freeze-dried microcapsules were evenly sprinkled onto the sample stage. Loose samples were gently blown away with a bulb syringe, ensuring the samples did not overlap. The sample stage was then placed in an ion sputtering deposition apparatus to deposit a gold film for 300 seconds. After deposition, the sample stage was placed in the sample chamber of the microscope. The voltage was set to 10 kV, the distance between the sample and the probe was approximately 8-10 mm, and a representative sample area was photographed at 500 X magnification to obtain the microstructure of the sample.

[0099] 4. Establishment of an iron deficiency anemia mouse model and evaluation of the effect of iron supplementation.

[0100] Six-week-old male Babl / c mice (weighing 22±2 g) were acclimatized for 7 days before a 7-day intestinal barrier injury modeling experiment was initiated. Mice in the model group were given 2.5% sodium dextran sulfate (DSS) solution instead of distilled water for 7 days, with the solution being changed every 2 days to induce an intestinal barrier injury model. Mouse weight was recorded on days 0, 3, and 7, and fecal and blood samples were collected. The Disease Activity Index (DAI) was calculated based on weight changes, fecal characteristics, and occult blood levels, and the content of lipoproteins in the feces was measured. The success of the model establishment was assessed by combining these indicators.

[0101] In the treatment intervention group, mice with DSS-induced intestinal barrier damage were randomly assigned to different groups. All groups were reinstated to drinking distilled water and underwent a 7-day intervention. The normal control group (NC) received no treatment and was fed routinely with free access to water. The model group (NE group) received 200 μL of distilled water per mouse daily by gavage; the clinical drug 5-ASA group received 200 μL of distilled water containing 1.2 mg mesalazine enteric-coated tablets per mouse daily by gavage; the 6% EGCG group received 200 μL of distilled water containing 1.2 mg EGCG powder per mouse daily by gavage; and the experimental group received 200 μL of distilled water containing 20 mg of the samples prepared in the various examples and comparative examples daily by gavage. All gavage solutions were freshly prepared and used immediately. After the intervention experiment, mice were fasted for 12 hours, and their weight changes were recorded. The mice were euthanized the following morning by cervical dislocation.

[0102] (1) Disease Activity Index (DAI)

[0103] According to the scoring criteria listed in the table below, mouse body weight, fecal characteristics, and fecal occult blood status were scored, and the Disease Activity Index (DAI) value was calculated based on these scores. The DAI score was calculated using a weighted cumulative score method, and the formula is as follows.

[0104] DAI = Weight change + Stool characteristics + Occult blood test

[0105] Table 1 Disease Activity Index (DAI) Scoring Table

[0106]

[0107] (2) Fecal lipoprotein (NGAL) detection

[0108] The level of NGAL in mouse feces was detected using the Elabscience mouse neutrophil gelatinase-associated lipocalin (NGAL) enzyme-linked immunosorbent assay (ELISA) kit. Before the experiment, the kit was equilibrated to room temperature. Fecal samples from each group of mice were collected, and standards, samples, and biotinylated antibodies were added sequentially according to the instructions. The samples were incubated at 37°C for 60 min and washed five times. Then, avidin-peroxidase complex was added, and the samples were incubated at 37°C for 30 min and washed again. The chromogenic solution was added, and the reaction was developed in the dark for 15 min. The reaction was terminated by adding 50 μL of stop solution. The absorbance of each well was measured at 450 nm using a microplate reader, and the concentration of NGAL in the samples was calculated based on the standard curve.

[0109] (3) In vivo fluorescence imaging

[0110] After successful modeling, colitis model mice were randomly grouped and administered the drug. Near-infrared fluorescence imaging was performed at 1, 2, 4, 6, and 24 hours post-administration using a small animal in vivo fluorescence imaging system. Mice were anesthetized with isoflurane inhalation before imaging, fixed in a prone position within the imaging chamber, with an excitation wavelength of 760 nm and an emission wavelength of 830 nm. The exposure time was automatically optimized based on signal intensity. After image acquisition, the fluorescence intensity in the abdominal intestinal region of the mice was quantitatively analyzed using the system's built-in software. Fluorescence intensity-time curves were plotted to compare the intestinal enrichment and retention capabilities of different formulations at different time points, and to evaluate the targeted delivery efficiency and in vivo distribution characteristics of the materials used in the examples and comparative examples.

[0111] (4) In vivo photoacoustic imaging

[0112] After successful modeling, normal mice and colitis model mice were administered the drug by gavage at a dose of 200 μL / mouse (with or without ICG concentration of 200 μg). Six hours after administration, the mice were anesthetized with isoflurane and fixed on the stage of the photoacoustic imaging system. The abdominal colon region was scanned. The excitation wavelengths were set to 780 nm, 808 nm, 820 nm and 840 nm, and the acquisition step size was 5 mm. Photoacoustic signal intensity, imaging resolution and target area signal distribution data were collected. Each group was scanned three times, and the average value was used for subsequent analysis.

[0113] (5) In vivo endoscopic imaging and microscopic imaging

[0114] Normal mice, colitis model mice, and mice after intervention were administered the drug (containing or without ICG concentration of 200 μL / mouse) via gavage. Six hours after administration, mice were anesthetized with 5% isoflurane, fixed supine on a 37°C heated imaging stage, and after perianal disinfection, the probe was coated with paraffin oil and inserted through the anus into the colonic region (depth 2-3 cm). Simultaneously, the microscopic imaging system was activated, and the fiber optic probe was positioned in the target area via a guide tube, maintaining a distance of 3-5 mm from the mucosa. Imaging parameters were set as follows: excitation wavelength 808 nm, scanning mode 360° rotation + axial translation (acquisition step size 5 mm), sampling frequency 200 MHz, ultrasound gain 40 dB, and an average signal intensity of 8 times per pixel. Signal intensity, imaging resolution, and target area signal distribution data were acquired, with each group scanned three times.

[0115] (6) Histological observation of important organs

[0116] After dissection of the mice, key organs such as the heart, liver, spleen, lungs, and kidneys were isolated. Each organ was placed in pre-cooled saline solution and gently rinsed to remove residual blood and connective tissue. The surface moisture was then blotted dry with sterile filter paper to ensure the tissue samples were clean. Tissue blocks from typical functional areas of each organ were immediately placed in 4% paraformaldehyde fixative and fixed at 4°C for 24-48 hours to ensure adequate tissue structure fixation. After fixation, the tissue blocks were processed according to standard paraffin embedding procedures: gradient dehydration (sequential immersion in 75%, 85%, 95%, and 100% ethanol), xylene clearing, and paraffin impregnation were performed before embedding the tissue blocks into paraffin blocks. The embedded blocks were cut into 5 μm thick continuous sections using a paraffin microtome. After spreading and retrieving the sections, they were baked in a 60°C oven for 2 hours to enhance adhesion between the sections and the glass slides. Hematoxylin-eosin (HE) staining was then performed: sections were dewaxed with xylene, rehydrated with graded ethanol, stained with hematoxylin for 5 min, differentiated with hydrochloric acid ethanol for 30 s, rinsed with running water for 10 min to regain blue color; then stained with eosin for 2 min, dehydrated with graded ethanol, cleared with xylene, and finally mounted with neutral resin. Tissue sections of various organs were observed using an optical microscope, and images were acquired at different fields of view (low magnification ×100, high magnification ×400).

[0117] Results analysis:

[0118] This invention's composite microcapsules achieve highly efficient co-encapsulation of EGCG and QMCC-ICG. Due to its low addition level of 1%, QMCC-ICG can fully utilize network binding sites without competing with EGCG, maintaining an encapsulation efficiency of nearly 100%. The encapsulation efficiency of EGCG also remains above 80%, regardless of its addition level. The EGCG-mediated physical cross-linking network constructed by the microcapsules demonstrates excellent gastrointestinal multi-barrier resistance and spatiotemporal controlled-release efficacy. In vitro digestion experiments show that the system achieves synergistic release of the two components from the terminal small intestine to the colon: the anti-inflammatory molecule EGCG has a release rate of <30% in the upper gastrointestinal tract, effectively avoiding drug loss in non-inflammatory areas and ensuring high-concentration delivery to colonic lesions. As shown in Table 3, the core photosensitizer probe QMCC-ICG achieves a targeted release rate of over 80% at 240 min, meeting the kinetic requirements of in situ colonic diagnosis and treatment.

[0119] Table 2. Encapsulation efficiency of different embodiments

[0120]

[0121] Table 3. Photosensitizer release rates in different embodiments

[0122]

[0123] like Figure 2 SEM results showed that the composite microcapsules in all examples formed a network structure with three-dimensional porous features, which provided a basis for loading active substances. Comparing the microstructures of the 6% EGCG-MC and 6% EGCG-MC@QMCC-ICG groups, the introduction of QMCC-ICG did not significantly damage the three-dimensional porous structure of the composite microcapsules, and its pore size and distribution characteristics were basically consistent with those of the EGCG-MC@QMCC-ICG group, indicating that the system has structural stability for co-loading multiple active ingredients.

[0124] (1) Dynamic monitoring of DAI scores

[0125] Dynamic monitoring of DAI scores was used to assess the impact of different intervention strategies on disease activity in mice with ulcerative colitis, such as... Figure 3As shown. After 7 days of intervention, the score of the normal group remained at a low level of 0.38±0.13. The score of the model group remained high (7.41±0.63). The scores of all example groups decreased significantly. Among them, Example 1 (6% EGCG-MC@QMCC-ICG) had a similar efficacy to 5-ASA (a first-line clinical drug), and the efficacy was not interfered with after the combination of optical probes, achieving synergy between imaging and targeted therapy. The score of the control group (0% EGCG-MC@QMCC-ICG) was similar to that of the model group, confirming that there is no intervention effect without EGCG. The efficacy of the free EGCG group at the same 6% dose was weaker, indicating that free polyphenols are easily degraded in the gastrointestinal tract and cannot establish an effective therapeutic concentration at the lesion, confirming the necessity of microcapsule encapsulation.

[0126] (2) Changes in fecal lipoprotein (NGAL) after drug intervention

[0127] like Figure 4 As shown, the NGAL content in the feces of normal mice was at an extremely low level (approximately 50 pg / mg), while it significantly increased in the model group to approximately 9600 pg / mg, indicating severe damage to the intestinal barrier. In Example 1 (6% EGCG-MC@QMCC-ICG) group, the NGAL content decreased significantly to approximately 2600 pg / mg, close to that of the comparative 6% EGCG-MC group (approximately 2700 pg / mg) and the 5-ASA group (approximately 2300 pg / mg), confirming that the barrier repair efficacy was not affected by the combined probe. The NGAL content in the comparative 0% EGCG-MC@QMCC-ICG group was similar to that of the model group, while the free 6% EGCG group had approximately 6000 pg / mg, further validating the crucial role of the microcapsule system in protecting and targeting the active ingredient.

[0128] (3) In vivo fluorescence imaging

[0129] like Figure 5 As shown, at a uniform dose of 20 mg / kg, the in vivo imaging performance of ICG, QMCC-ICG, and 6%EGCG-MC@QMCC-ICG was compared. The 6%EGCG-MC blank carrier group showed no signal throughout, excluding autofluorescence interference. The free ICG group showed extremely high signal at 1 h but a sharp decline at 2 h, revealing its rapid degradation by gastric acid and short half-life. The QMCC-ICG group maintained fluorescence levels of 1.8–2.1 × 10⁻⁶ h from 1 to 6 h. 9 p / s / cm 2 / s, demonstrating that QMCC loading effectively improves ICG stability, but due to the lack of outer layer embedding, the targeting retention efficiency is insufficient. The 6% EGCG-MC@QMCC-ICG group showed a low signal at 1 h (approximately 1.3 × 10⁻⁶). 9 p / s / cm 2The signal strength was 10.5 × 10⁶ / s, indicating that the outer microcapsule effectively shielded ICG in the stomach; the signal peaked at 6 h (approximately 10.5 × 10⁶ / s). 9 p / s / cm 2 The signal density ( / s) indicates that the probe is highly concentrated at the site of colon inflammation; the signal is significantly reduced after 24 hours, demonstrating good in vivo metabolic clearance ability.

[0130] (4) Endoscopic imaging analysis

[0131] Photoacoustic endoscopic imaging (PAE) technology was used to visually assess intestinal vascular network remodeling after intervention, such as... Figure 6 As shown, the vascular network of the intestinal wall in normal mice is sparse and regular, while in UC model mice, the blood vessels are extremely dense and abnormally congested, resulting in extremely strong photoacoustic signals. Although the free ICG group improved the overall photoacoustic signal, the lack of targeted anchoring ability and non-specific leakage led to blurred vascular boundaries and severe background interference, making it difficult to accurately reflect the vascular morphology of the lesions. In contrast, after intervention with 6% EGCG-MC@QMCC-ICG in Example 1, the number of abnormal high-signal lesions in the intestine was significantly reduced, and the vascular density and signal intensity decreased significantly, approaching the normal state; the vascular boundaries were clear and sharp, indicating that the photosensitizer QMCC-ICG achieved precise targeted delivery to the inflammatory site, and the released polyphenols synergistically inhibited abnormal angiogenesis. This result confirms the therapeutic value of the composite microcapsules in promoting the reversal of vascular pathology while maintaining excellent imaging performance.

[0132] (5) Microscopic imaging analysis

[0133] Photoacoustic microscopy (PAM) was used to assess microcirculatory hemodynamics at micrometer-level resolution, and pseudo-color images of the intestinal microvascular network were obtained for different treatment groups, such as... Figure 7 As shown in the PAM images, the model group mice exhibited disordered capillary beds with small abnormal high-signal foci, reflecting inflammation-driven microcirculatory disturbances; while the normal mouse capillary beds were highly ordered with lower signal levels. The comparative free ICG group showed severe background noise due to nonspecific leakage, resulting in poor contrast between diseased microvessels and normal tissue, and insufficient targeting. In contrast, the 6% EGCG-MC@QMCC-ICG group in Example 1 demonstrated a high signal-to-noise ratio, with effective suppression of background signals, and clear identification of abnormally dilated capillaries and congestion foci; after intervention, the degree of capillary network disorder was significantly reduced, and the vascular morphology tended to be more regular. This improvement stems from a dual mechanism: the QMCC carrier utilizes the high permeability of the damaged mucosa to achieve deep probe penetration and high-contrast imaging; the released EGCG blocks the pro-angiogenic pathway, reducing abnormal microvascular angiogenesis and increased permeability. The PAM results confirm that this material possesses both high-resolution lesion localization and microcirculation repair efficacy.

[0134] To systematically investigate the toxicological characteristics of different composite microcapsules in vivo, H&E staining and histopathological analysis were performed on five important solid organs (heart, liver, spleen, lung, and kidney) of mice in each group. Figure 8 The results showed that, compared with the normal group (NC), the model group (NE) mice exhibited slight scattered vacuolation in the liver and mild congestion in the lungs. These minor histological changes are attributed to the systemic metabolic and immune stress response induced by ulcerative colitis (UC) and did not constitute substantial organ damage, thus providing a baseline for subsequent evaluation of the safety of exogenous intervention materials. The histological morphology of all organs remained highly intact after intervention in all samples. This result confirms that the oxidized starch and gelatin complex polysaccharide carrier system based on QMCC-ICG photosensitizer and mediated by EGCG possesses excellent in vivo biocompatibility, effectively exerting local therapeutic effects on the colon without inducing any systemic toxicity.

[0135] In summary, the composite microcapsules constructed in this invention successfully achieve integrated diagnosis and treatment of ulcerative colitis, combining precise targeted imaging with efficient anti-inflammatory repair. This demonstrates their excellent biocompatibility and reveals their synergistic effect across multiple mechanisms. This research provides new design ideas for the development of integrated oral-colonic targeted diagnostic and therapeutic materials, possessing significant scientific and application value.

[0136] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a colon-targeted diagnostic and therapeutic integrated microcapsule material, characterized in that, Includes the following steps: (1) Modified cellulose with opposite charges is mixed and reacted with photosensitizer to obtain cellulose-photosensitizer complex with electrostatic interaction; (2) Heat the oxidized starch to gelatinize it and mix it with the gelatin solution. Then add the cellulose-photosensitizer complex and polyphenol aqueous solution from (1), stir thoroughly, and finally let it stand to obtain the microcapsules that have been preliminarily shaped. (3) The microcapsules that have been preliminarily shaped are placed under refrigeration conditions for sufficient aging and shaping, and then vacuum freeze-dried to obtain the integrated microcapsule material for colon-targeted diagnosis and treatment.

2. The preparation method according to claim 1, characterized in that, The concentration of the gelatin solution in step (2) is 5% to 15% w / v; the mass ratio of oxidized starch to gelatin is 1:100 to 100:

1.

3. The preparation method according to claim 1, characterized in that, The oxidized starch mentioned in step (2) is carboxylated starch with a carboxyl content of 0.01% to 0.5%; the polyphenol content is 0.1% to 20% w / v of the dry weight of starch.

4. The preparation method according to claim 1, characterized in that, The cellulose-photosensitizer complex mentioned in step (2) accounts for 0.1% to 10% of the total dry weight of starch.

5. The preparation method according to claim 1, 2, 3, or 4, characterized in that, The modified cellulose in step (1) is a positively charged cellulose obtained by cationization modification with quaternary ammonium salt; the photosensitizer is a negatively charged photosensitizer; the mass ratio of the photosensitizer to the modified cellulose is 1:100 to 1:

1.

6. The preparation method according to claim 5, characterized in that, The cellulose mentioned in step (1) is cellulose derived from wood pulp or cotton pulp, with an average particle size of 20 nm to 200 μm; the photosensitizer is indocyanine green. The polyphenols in the polyphenol aqueous solution in step (2) are selected from at least one of tannic acid, epigallocatechin gallate, proanthocyanidins, gallic acid, and tea polyphenols; the gelatin is type A gelatin or type B gelatin. The oxidized starch mentioned in step (2) is oxidized starch obtained by oxidizing starch raw materials; the starch raw materials are selected from at least one of corn starch, potato starch, cassava starch and other starches; the oxidizing agent for the oxidation treatment is at least one of sodium periodate, sodium hypochlorite and hydrogen peroxide.

7. The preparation method according to claim 1, 2, or 3, characterized in that, The mixing reaction described in step (1) is carried out under light-protected conditions, with stirring for 4 to 12 hours at pH 6 to 10 and temperature 0 to 55°C, and the concentration of metal salt in the reaction system is 0 to 20 mg / L; The heating and gelatinization conditions described in step (2) are as follows: disperse oxidized starch in water, stir and gelatinize at 80-95°C for 20-60 minutes, and then cool to room temperature; The refrigeration conditions in step (3) are: temperature 2℃~8℃, refrigeration time 12~48 hours; the vacuum freeze-drying conditions are: temperature -50℃~-80℃, vacuum degree less than 10 Pa, drying time 24~72 hours.

8. The preparation method according to claim 7, characterized in that, The pH value of the mixed reaction in step (1) is 7-9, the temperature is 15-35℃, and the sodium chloride concentration is 0-10 mg / L; the preparation of the modified cellulose is as follows: cellulose is swollen in an alkali metal hydroxide solution, and then 2,3-epoxypropyltrimethylammonium chloride is added as a quaternizing agent. The reaction is carried out at 20-80℃ for 1-5 hours. After neutralization, washing and drying, the modified cellulose is obtained.

9. The colon-targeted diagnostic and therapeutic integrated microcapsule material prepared by the method according to any one of claims 1 to 8, characterized in that, The microcapsule material comprises a wall material composed of an oxidized starch-gelatin-polyphenol crosslinking network, and a cellulose-photosensitizer complex dispersed in the wall material.

10. The use of the colon-targeted diagnostic and therapeutic integrated microcapsule material of claim 9 in the preparation of products for diagnosing and treating colon diseases.

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