A colon targeting delivery system, its preparation method and use

Abstract

A colon-targeted delivery system, a preparation method and application thereof, comprising amifostine, pectin and chitosan; through a chemical amidation reaction, carboxyl in the pectin and amino in the amifostine are connected through an amide bond to form a nanoparticle core PEC-AMF, and the chitosan and the pectin form a nanoparticle coating layer through electrostatic interaction to synthesize CS / PEC-AMF nanoparticles; the colon-targeted delivery system in the application realizes precise and controlled drug release by means of the step-by-step change of pH values in the gastrointestinal tract environment and the catalysis of pectinase in the colon; experiments prove that the delivery system has excellent stability in the stomach and small intestine, and can rapidly release amifostine in the colon, thereby effectively protecting the intestinal epithelium and immune cells from damage caused by radiotherapy, while the pectin regulates the intestinal microecology, protects the normal tissues of the colorectum and does not affect the effect of radiotherapy on tumors.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and nanomedicine delivery technology, and in particular to a colon-targeted delivery system, its preparation method, and its application. Background Technology

[0002] In recent years, the incidence of colorectal cancer has been rising year by year, becoming one of the most serious malignant tumors threatening human health worldwide. Radiotherapy, as an important treatment for colorectal cancer and other abdominal and pelvic tumors, has achieved significant efficacy in killing tumor cells, but it also inevitably damages the surrounding normal colorectal tissue. Radiation-induced apoptosis of intestinal epithelial cells, DNA double-strand breaks, intestinal barrier disruption, and inflammatory responses often lead to acute radiation enteritis and long-term intestinal dysfunction, severely affecting patients' quality of life and potentially inducing secondary intestinal malignancies.

[0003] Amifostine (AMF), a clinically recognized radioprotective agent, is widely used in radiotherapy to reduce the side effects on normal tissues because it can be selectively converted into a reactive free radical scavenger in normal cells, effectively reducing the generation of oxygen free radicals and cell damage caused by radiation. However, AMF has significant limitations: when administered orally, it is rapidly degraded in the upper gastrointestinal tract by gastric acid and digestive enzymes, resulting in a significantly reduced drug concentration by the time it reaches the colorectal region, thus failing to exert its protective effect. Although intravenous injection can improve the stability and bioavailability of AMF to some extent, this method of administration is inconvenient, prone to causing systemic adverse reactions, and unsuitable for radiotherapy protection in the colorectal region.

[0004] In view of the above problems, there is an urgent need to develop a novel drug delivery system that can effectively protect AMF from degradation in the upper gastrointestinal tract via oral administration and achieve its targeted release in the colorectal region. Summary of the Invention

[0005] The purpose of this invention is to provide a colon-targeted delivery system, its preparation method, and its application. This system utilizes nanoparticles constructed from prebiotic pectin and chitosan to achieve precise release of amifostine in the colon through a dual response mechanism of pH changes and enzymatic degradation by intestinal flora. Simultaneously, it repairs the intestinal microecological environment, thereby effectively protecting the colonic region, reducing the risk of secondary tumors, and improving the patient's quality of life without affecting the efficacy of tumor treatment, thus solving the problems existing in the prior art.

[0006] A colon-targeted delivery system comprising amifostine, pectin, and chitosan;

[0007] The carboxyl groups in the pectin and the amino groups in the aminophosphine are linked by amide bonds to form the nanoparticle core PEC-AMF. The chitosan and pectin form a nanoparticle coating layer through electrostatic interaction, thus synthesizing CS / PEC-AMF nanoparticles.

[0008] The CS / PEC-AMF nanoparticles synthesized in this invention remain stable in the stomach and small intestine after oral administration, and achieve precise drug release in the colon in response to changes in environmental pH and pectinase catalysis.

[0009] Preferably, the CS / PEC-AMF nanoparticles are spherical and have an average particle size of 350–400 nm.

[0010] Preferably, the purity of the amifostine is ≥99.0%;

[0011] The pectin contains ≥74.0% galacturonic acid with a molecular weight of 20,000-40,000 Da;

[0012] The chitosan has a degree of deacetylation ≥ 75.0% and a viscosity of 20–300 mPas.

[0013] Preferably, it also includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and triethylamine as crosslinking agents.

[0014] In this invention, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, used as a crosslinking agent, promotes the formation of amide bonds, thereby promoting the amidation reaction of pectin with aminophosphine and stabilizing the PEC-AMF structure. Triethylamine is used to adjust the pH of the reaction solution.

[0015] The pectin molecule of this invention has a main chain composed of α-1,4-D-galacturonic acid units, and the side chains contain different neutral sugar residues. Therefore, the pectin molecule contains a large number of free carboxyl groups, hydroxyl groups and other functional groups that can be used for molecular modification or drug linkage.

[0016] Using 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) as a catalyst, the carboxyl groups in pectin molecules are activated. Amifostine then attaches to the pectin molecule backbone through amide bonds formed between the amino groups in the molecule and the activated carboxyl groups, forming an amifostine-pectin graft (PEC-AMF), thereby improving the stability of amifostine in the upper gastrointestinal tract after oral administration.

[0017] This invention discloses a method for preparing a colon-targeted delivery system, characterized by comprising the following steps:

[0018] S1: Preparation of PEC-AMF nanoparticle core

[0019] S1.1: Dissolve pectin in distilled water and stir to form solution A;

[0020] S1.2: Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to solution A and stir to form solution B;

[0021] S1.3: Triethylamine and amifostine were added to solution B, stirred, dialyzed, and freeze-dried to obtain PEC-AMF nanoparticles;

[0022] Step 2: Preparation of CS / PEC-AMF nanoparticles

[0023] S2.1: Weigh the PEC-AMF nanoparticles synthesized in S1.3 and dissolve them in distilled water. Adjust the pH of the solution to form solution C.

[0024] S2.2: Dissolve chitosan in distilled water, adjust the pH value, and form solution D;

[0025] S2.3: Add solution D to solution C, stir, filter, and obtain CS / PEC-AMF nanoparticles.

[0026] Preferably, characterization and testing of CS / PEC-AMF nanoparticles;

[0027] The particle size and dispersion index of nanoparticles were determined using dynamic light scattering instrumentation, the morphology of nanoparticles was observed using transmission electron microscopy, and the drug loading and encapsulation efficiency of amifostine were determined by high performance liquid chromatography.

[0028] Preferably, in S1.2, the amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1.5 to 2 times the molar amount of carboxyl groups in the pectin;

[0029] In S1.3, the amount of triethylamine used is 1.2 to 1.5 times the molar amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

[0030] The dosage of amifostine is 0.5 to 1 times the molar amount of carboxyl groups in pectin.

[0031] Preferably, in S2.1, the mass ratio of PEC-AMF nanoparticles to distilled water is 1:20 to 1:50;

[0032] In S2.2, the chitosan concentration is 0.5–1.5% w / v;

[0033] In S2.3, the mass ratio of pectin to chitosan is 2:1.

[0034] Preferably, in S1.1, the stirring rate is 300-600 rpm and the stirring time is 10-25 min;

[0035] In S1.2, the stirring rate is 300–600 rpm, the stirring time is 10–20 min, and the pH range is 4.5–6.0. ​​This maintains the activity of the carboxyl group, and the amidation reaction requires thorough stirring. Dialysis is used to remove unreacted substances.

[0036] In S1.3, the stirring time is 24 hours, the stirring rate is 300-600 rpm, and the dialysis conditions are: the molecular weight cutoff value of the dialysis bag is 6,000-8,000 Da, the dialysis time is 48 hours, and the water is changed at least 3 times.

[0037] In S2.2, the rate of solution addition is 1-2 mL / min, the pH value is 4.5-5.5, the stirring rate is 300-500 rpm, the stirring time is 30 minutes or until completely dissolved, and the temperature is 25-40℃.

[0038] In S2.3, the feeding rate is 1-2 mL / min (using titration), the stirring rate is 300-500 rpm, and the stirring time is 12-24 h.

[0039] Preferably, in step S2.3, a polyvinylidene fluoride or nylon filter membrane with a pore size of 0.22 μm is selected.

[0040] Preferably, the present invention also discloses the application of the colon-targeted delivery system in the preparation of intestinal radiotherapy drugs.

[0041] The drug prepared by this invention can maintain stability in the stomach and small intestine, and achieve precise drug release in the colon in response to changes in environmental pH and the catalytic action of pectinase, thereby effectively protecting the intestinal epithelium and immune cells, regulating the intestinal microecology, protecting normal colorectal tissues without affecting the effect of radiotherapy on tumors.

[0042] This invention discloses a colon-targeted delivery system. The core of this system is a dual-responsive nanoparticle constructed from aminofistine, pectin, and chitosan, which has the following technical effects:

[0043] (1) This dual-responsive delivery system utilizes its stability in gastric and intestinal fluids to prevent premature release of amifostine after oral administration. When the nanoparticles reach the colon region, the pectin is partially degraded due to the increase in local pH and the catalytic action of pectinase unique to the colon, which induces the destruction of the nanoparticle structure, thereby achieving precise and controlled release of amifostine. This not only significantly increases the local drug concentration in the colon and enhances the protective effect against radiation-induced colorectal damage, but also further alleviates the damage to the intestines caused by radiotherapy by repairing the intestinal microecology, while reducing the risk of systemic adverse reactions and improving patient compliance and quality of life.

[0044] (2) This system utilizes dual-responsive nanoparticles constructed from pectin and chitosan to ensure that amifostine remains stable in the stomach and small intestine after oral administration, thus avoiding degradation of the drug in the upper digestive tract due to acidic environment and digestive enzymes, thereby significantly improving the bioavailability of the drug.

[0045] (3) When the nanoparticles enter the colon after passing through the gastrointestinal tract, they respond to the increase in local pH and the catalytic action of pectinase unique to the colon, achieving precise and controlled release of amifostine. This targeted drug release mechanism ensures that the drug reaches a high local concentration in the colon, thereby effectively protecting the colorectal epithelium and immune cells and reducing the risk of local damage caused by radiotherapy.

[0046] (4) The system of the present invention fully utilizes the prebiotic properties of pectin and chitosan, which can promote the growth of beneficial bacteria and improve and repair the damaged intestinal microecological environment. This not only helps to enhance the intestinal barrier function, but also regulates intestinal immunity to a certain extent and reduces the risk of secondary tumors.

[0047] (5) The nanoparticles of the present invention are administered orally, which has the advantages of simple operation and high patient compliance, avoiding the systemic adverse reactions and operational inconvenience caused by traditional injection administration, and further improving the safety and comfort of clinical application. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0049] Figure 1 : Schematic diagram of the preparation of CS / PEC-AMF in this invention;

[0050] Figure 2 The synthetic route of PEC-AMF in this invention;

[0051] Figure 3 : NMR spectra of PEC-AMF synthesized in this invention;

[0052] Figure 4 TEM image of CS / PEC-AMF constructed in this invention;

[0053] Figure 5 : Schematic diagram and TEM image changes of CS / PEC-AMF in response to pH and pectinase in this invention for releasing AMF;

[0054] Figure 6The present invention provides a map showing the content of AMF in different parts of the intestine after oral administration of CS / PEC-AMF to mice.

[0055] Figure 7 The present invention describes the changes in the intestinal tract of mice after oral administration of CS / PEC-AMF and subsequent abdominal and pelvic irradiation.

[0056] Figure 8 The present invention describes the changes in key indicators of intestinal epithelial cells in mice after oral administration of CS / PEC-AMF and subsequent abdominal and pelvic irradiation.

[0057] Figure 9 The present invention is a map showing the changes in intestinal immune cells in mice after oral administration of CS / PEC-AMF and subsequent abdominal and pelvic irradiation.

[0058] Figure 10 The present invention describes the changes in the intestinal flora of mice after oral administration of CS / PEC-AMF and subsequent abdominal and pelvic irradiation.

[0059] Figure 11 The present invention describes the changes in intestinal tissue in mice after oral administration of CS / PEC-AMF and repeated abdominal and pelvic radiation to simulate long-term injury.

[0060] Figure 12 The present invention presents a chromatogram of the therapeutic effects of oral administration of CS / PEC-AMF followed by abdominal and pelvic irradiation in mice with colon cancer.

[0061] Figure 13 This invention presents an atlas of changes in normal intestinal tissue in mice with colon cancer after oral administration of CS / PEC-AMF followed by abdominal and pelvic radiation therapy.

[0062] Figure 14 The present invention presents a chromatogram of the therapeutic effects of oral administration of CS / PEC-AMF followed by abdominal and pelvic irradiation in mice with rectal cancer.

[0063] Figure 15 This invention presents an atlas of changes in normal intestinal tissue in mice with rectal cancer after oral administration of CS / PEC-AMF followed by abdominal and pelvic radiation therapy.

[0064] Figure 16 The protective effect of oral administration of CS / PEC-AMF to mice against radiation-induced intestinal secondary tumors is illustrated in the graph. Detailed Implementation

[0065] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0066] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0067] In recent years, nanoparticle systems based on natural polysaccharide materials have gradually become a research hotspot. Among them, pectin, due to its acid resistance and ability to be degraded by specific microbial enzymes in the colon, is considered an ideal material for constructing colon-targeting carriers. Chitosan, on the other hand, possesses excellent biocompatibility and stable encapsulation capabilities, enabling it to form a tight bond with the pectin-amyfifine complex, thus constructing a nanoparticle system with dual responsiveness (i.e., pH response and microbial enzyme response). This system remains stable in gastric and intestinal fluids, but once it enters the colorectal region, under higher pH values ​​and specific enzymatic action, the pectin portion degrades, thereby triggering the site-specific release of AMF, increasing local drug concentration, enhancing protective efficacy, and reducing systemic side effects.

[0068] Therefore, this invention proposes a colon-targeted delivery system, its preparation method, and its application, which has dual responsiveness. It aims to overcome the gastrointestinal degradation problem during oral administration by rationally modifying AMF, so as to achieve efficient and targeted release in the colorectal region, thereby effectively alleviating colorectal damage caused by radiotherapy, while ensuring the therapeutic efficacy against tumors.

[0069] Example 1:

[0070] A method for preparing a colon-targeted delivery system includes the following steps:

[0071] Step 1: Preparation of PEC-AMF nanoparticle core

[0072] like Figure 2 As shown, firstly, pectin with a galacturonic acid content of not less than 74.0% and a molecular weight of 30,000 Da is selected, dissolved in an appropriate amount of distilled water, and stirred at a stirring speed of 500 rpm for 15 minutes to form a uniform solution A.

[0073] Then, EDC·HCl was added to solution A, the amount of which was calculated as twice the molar amount of carboxyl groups in the pectin. At the same time, the reaction was carried out at a stirring rate of 500 rpm for 15 minutes, and the pH of the reaction solution was adjusted to 5 to maintain the activity of the carboxyl groups. At this time, solution B was formed.

[0074] Next, triethylamine, which is 1.5 times the molar amount of EDC·HCl, and aminofristine, which is 1 times the molar amount of carboxyl groups in pectin, were added to solution B in sequence. The mixture was stirred thoroughly for 24 hours at the same 500 rpm to promote the formation of amide bonds between the amino groups in aminofristine molecules and the carboxyl groups in pectin molecules, thereby generating a stable pectin-aminofristine complex, namely PEC-AMF.

[0075] Subsequently, the reaction mixture was placed in a dialysis bag with a molecular weight cutoff of 8000 Da for dialysis for 48 hours, during which the water was changed 3 times to remove unreacted substances and low molecular weight impurities.

[0076] Finally, the dialysis solution was freeze-dried to obtain pure and stable PEC-AMF nanoparticles.

[0077] Figure 3 The nuclear magnetic resonance (NMR) spectrum of PEC-AMF after synthesis was shown to confirm the formation of amide bonds. The results showed that the characteristic peaks of AMF and pectin were identified in the 1H NMR spectrum, and the shift of the AMF peak indicated the formation of amide bonds, which indicated that the synthesis of PEC-AMF was successful.

[0078] Step 2: Preparation of CS / PEC-AMF nanoparticles

[0079] like Figure 1 As shown, an appropriate amount of the PEC-AMF nanoparticles obtained in step 1 is weighed and dissolved in distilled water. The mass ratio of PEC-AMF to distilled water is preferably controlled within the range of 1:30. The pH of the solution is adjusted to 5.0 by adding an appropriate amount of acid or alkali to form solution C.

[0080] Meanwhile, chitosan was dissolved in distilled water at a concentration of 1% (w / v), and the pH of the solution was adjusted to 5.0 to form solution D;

[0081] Subsequently, solution D was slowly added to solution C at a rate of 1 mL / min using a dropwise method. The mixture was stirred at 500 rpm for 24 hours at room temperature to allow chitosan and PEC-AMF to fully combine through electrostatic interaction, forming uniform chitosan-coated PEC-AMF nanoparticles, namely CS / PEC-AMF.

[0082] The filtration step involves filtering the reaction mixture using a 0.22 μm filter membrane to remove unbound or aggregated particles, thus obtaining the final product.

[0083] Figure 4 The transmission electron microscope (TEM) images show the spherical morphology of the prepared nanoparticles, with particle sizes between 350 and 400 nm.

[0084] A colon-targeted delivery system obtained by the above preparation method includes aminofristine, pectin, and chitosan;

[0085] The carboxyl groups in pectin and the amino groups in aminophosphine are linked by amide bonds to form the nanoparticle core PEC-AMF. Chitosan and pectin form a nanoparticle coating layer through electrostatic interaction, thus forming CS / PEC-AMF nanoparticles.

[0086] In this invention, amifostine is a radioprotective agent, pectin is a prebiotic and encapsulating agent, and chitosan is a film-forming material for nanoparticles.

[0087] In this embodiment, the nanoparticles are spherical with a particle size of 372 nm; they have good dispersibility and stability; the chitosan coating provides a physical barrier to ensure the stability of the nanoparticles in the stomach and small intestine, while ensuring drug release is triggered by the dual stimulation of pH and gut microbiota in the colonic environment.

[0088] Physical properties

[0089] Particle size distribution: The average particle size was 372±35.5 nm and the PDI (polydispersity index) was 0.154, as determined by dynamic light scattering (DLS), indicating that the particles have good uniformity.

[0090] Potential: The zeta potential of the nanoparticles is -8.54±1.13mV, indicating that the particles have a certain surface charge, which ensures their stability in solution.

[0091] Drug loading: The drug loading of amifostine was determined to be 13.7 ± 1.7% by high performance liquid chromatography (HPLC), ensuring effective release of the drug at the target site.

[0092] pH / enzyme dual responsiveness: The nanoparticles are stable in simulated gastric juice (pH 2.0) and small intestinal juice (pH 6.8), while rapidly releasing the drug in simulated colonic juice (pH 7.4), exhibiting good multi-response properties.

[0093] This invention constructs nanoparticles with multi-responsive properties through the amidation reaction of pectin and aminofistin, and by using chitosan as a coating material.

[0094] In this embodiment, the purity of amifostine is ≥99.0%;

[0095] Pectin contains ≥74.0% galacturonic acid with a molecular weight of 20,000-40,000 Da;

[0096] Chitosan has a degree of deacetylation ≥75.0% and a viscosity of 20-300 mPas.

[0097] In this embodiment, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and triethylamine are also included as crosslinking agents. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride promotes the amidation reaction of pectin and aminofistin. Triethylamine is used to adjust the pH of the reaction solution.

[0098] In vitro dual-response drug release experiment

[0099] To verify the dual-responsive drug release performance of CS / PEC-AMF nanoparticles, the prepared nanoparticles were placed in simulated gastric fluid (pH 1.5–3.5), simulated small intestinal fluid (pH 5.5–6.8), and simulated colonic fluid (pH 6.4–7.8, containing collagenase) for in vitro drug release tests.

[0100] Test results showed that the nanoparticles maintained good stability in simulated gastric and small intestinal fluids, with extremely low AMF release; however, in simulated colonic fluid, due to the higher pH and the catalytic effect of pectinase, the nanoparticles rapidly underwent structural destruction, resulting in a large release of AMF. Figure 5 As shown, the diagram illustrates the response release and TEM image changes, comparing the morphological changes and drug release of nanoparticles under different environments, demonstrating their ability to release drugs at specific points in the colon region.

[0101] Distribution in animal bodies

[0102] After oral administration of CS / PEC-AMF nanoparticles to mice, the AMF content in the intestinal tissue of the mice was detected at different time points using airflow-assisted desorption / electrospray ionization mass spectrometry (AFADESI-MSI) to detect frozen sections of the entire gastrointestinal tract and obtain representative mass signals (m / z 135.0945, corresponding to the active form of AMF). Figure 6 As shown in the figure, 6 hours after oral administration, the AMF signal in the colon region of the CS / PEC-AMF nanoparticle group was significantly higher than that of the free AMF or PEC-AMF group. LC-MS analysis further confirmed the efficient accumulation of AMF in the colon, thus demonstrating the colon-targeted drug release advantage of this system.

[0103] Evaluation of acute radiation-induced intestinal injury model

[0104] An acute radiation injury model was established using healthy C57BL / 6 mice. Six hours after oral administration of CS / PEC-AMF nanoparticles (AMF dose 200 mg / kg), mice were irradiated with X-rays (dose 12 Gy) throughout the abdomen. Three days post-irradiation, mice were sacrificed, and colorectal tissue was collected to directly investigate the extent of damage by observing colorectal length. Immunofluorescence staining was performed on key intestinal epithelial cell parameters, and immunofluorescence staining was also used to observe changes in intestinal macrophages. Finally, 16S rRNA sequencing analysis was performed on the intestinal flora of mice following oral administration of CS / PEC-AMF and subsequent abdominal and pelvic irradiation.

[0105] The results are as follows Figure 7 As shown, the irradiated group exhibited significant intestinal tissue damage and a marked shortening in length, while the CS / PEC-AMF protection group showed the least change in intestinal length, most closely resembling the intestinal characteristics of normal mice. Simultaneously, Lysozyme, Mucin2, and Lgr5 were used as markers to evaluate the expression of Paneth cells, goblet cells, and intestinal stem cells in the intestine after radiation injury.

[0106] The results are as follows Figure 8 As shown, after irradiation, the fluorescence signals of Lysozyme and Mucin2 were significantly weakened, indicating a reduction in the number of intestinal secretory cells (including Paneth cells and goblet cells) induced by radiation. At the same time, the number of Lgr5-positive cells was significantly reduced, suggesting damage to intestinal stem cells. Conversely, after protection with CS / PEC-AMF nanoparticles, the expression of Lysozyme and Mucin2 was significantly restored, and the fluorescence signal was close to the control group level. Meanwhile, the number of Lgr5-positive cells also increased significantly, indicating that nanoparticles can effectively protect and repair damaged intestinal secretory cells and stem cells, promote the regeneration and repair of intestinal epithelium, and improve intestinal barrier function.

[0107] The results are as follows Figure 9 As shown, after radiation treatment, the number of CD86-positive cells (a marker of M1 macrophages) in intestinal tissue significantly increased, while the number of CD206-positive cells (a marker of M2 macrophages) significantly decreased, indicating that radiation promoted the dominance of pro-inflammatory M1 macrophages. After treatment with CS / PEC-AMF nanoparticles, the expression level of CD86 in the intestine significantly decreased, while the number of CD206-positive cells significantly increased, suggesting that this treatment significantly promoted the transformation of macrophages from M1 to M2 types, thereby exerting anti-inflammatory and tissue repair effects. This transformation helps restore intestinal immune balance and alleviate radiation-induced inflammatory damage.

[0108] The results are as follows Figure 10As shown, the α-diversity of gut microbiota reflected a significant decrease in the α-diversity of gut microbiota in irradiated mice, indicating that radiation caused a decline in gut microbiota diversity. Compared with the irradiated group, the α-diversity of gut microbiota in mice orally administered CS / PEC-AMF nanoparticles was significantly restored, showing that this treatment can effectively repair the impact of radiation damage on gut microbiota and increase the diversity and stability of the microbiota. β-diversity reflected a significant difference between the gut microbiota of irradiated mice and the normal group, indicating that radiation significantly altered the structure of gut microbiota. After treatment with CS / PEC-AMF nanoparticles, the β-diversity of gut microbiota was significantly improved, indicating that this treatment can effectively restore the structure of gut microbiota, bringing it closer to a normal state, and further supporting its function in repairing gut microbiota.

[0109] Evaluation of a chronic radiation-induced intestinal injury model

[0110] A long-term radiation chronic injury model was established using healthy C57BL / 6 mice. After oral administration of CS / PEC-AMF nanoparticles, intestinal injury was evaluated 30 days after abdominal and pelvic radiation, as described in Example 5. Results are as follows: Figure 11 As shown, compared with the irradiation-only group, the intestinal tissue length and histopathological evaluation of the CS / PEC-AMF treatment group were significantly closer to those of normal mice, indicating that the system can effectively reduce the degree of chronic intestinal damage caused by long-term radiation.

[0111] Protective effect of CS / PEC-AMF during radiotherapy in mice with colon cancer

[0112] In a colon cancer model, an orthotopic colon cancer mouse model was used for evaluation. After establishing the colon cancer model, mice were orally administered CS / PEC-AMF nanoparticles (AMF dose 200 mg / kg), and 6 hours later received abdominopelvic radiotherapy (12 Gy whole abdomen irradiation). After radiotherapy, changes in tumor volume were monitored periodically by bioluminescence imaging, and colorectal tissue was harvested at the time of treatment completion to observe tumor bearing.

[0113] The results are as follows Figure 12 As shown, the tumor volume in mice treated with CS / PEC-AMF decreased significantly, indicating that the nanoparticles enhanced the tumor-suppressing effect in combination with radiotherapy; while Figure 13 The results showed that the colorectal tissue in this group carried fewer tumors, suggesting that the nanoparticles had a protective effect against radiation damage, while not interfering with the tumor-killing effect of radiotherapy.

[0114] Protective effect of CS / PEC-AMF during radiotherapy in mice with rectal cancer

[0115] In a rectal cancer model, an in vivo mouse model of rectal cancer was used for evaluation. Mice were established for rectal cancer by rectal injection of SW620 cells, followed by oral administration of CS / PEC-AMF nanoparticles (same dose and administration regimen) and intra-abdominal pelvic radiotherapy. Tumor growth dynamics were monitored during and after radiotherapy using bioluminescence imaging.

[0116] The results are as follows Figure 14 As shown, the growth of rectal tumors in the CS / PEC-AMF treatment group was significantly inhibited; simultaneously, observation of tumor-bearing normal rectal tissue revealed that it bore fewer tumors. Figure 15 As shown, this demonstrates that nanoparticles maintain the effective inhibitory effect of radiotherapy on tumors while protecting normal rectal tissue.

[0117] Effects of radiotherapy on intestinal tissue during secondary colorectal cancer

[0118] To evaluate the role of CS / PEC-AMF nanoparticles in reducing the risk of secondary intestinal tumors after radiotherapy, a secondary tumor model was established. Colon-bearing mice were subjected to intra-abdominal pelvic radiation, and the incidence of secondary intestinal tumors was statistically analyzed over a long period. Results are as follows: Figure 16 As shown, mice treated with CS / PEC-AMF nanoparticles orally had a significantly lower incidence of secondary intestinal tumors than the control group, suggesting that the system not only plays a protective role in acute and chronic radiation injury, but also effectively reduces the risk of radiation-induced secondary malignant lesions.

[0119] This invention synthesizes a dual-responsive nanoparticle colon-targeted delivery system based on amifostine, pectin, and chitosan. This system utilizes the unique properties of natural materials (pectin and chitosan) to achieve dual-responsive drug release in vivo through responses to the specific pH environment of the colonic region and the enzymatic degradation by intestinal microorganisms, thereby achieving precise drug delivery to the colonic region. This technology is applicable to the treatment of colon-related diseases and can also be used in radiotherapy to protect colonic tissue from radiation damage.

[0120] It should also be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0121] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

[0122] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A colon-targeted delivery system, characterized in that, Amifostine, pectin and chitosan; The carboxyl in the pectin and the amino in the amifostine are connected by an amide bond to form a nanoparticle core PEC-AMF, and the chitosan and the pectin are connected by electrostatic interaction to form a nanoparticle coating layer, thereby synthesizing CS / PEC-AMF nanoparticles.

2. The colon targeted delivery system according to claim 1, wherein: The CS / PEC-AMF nanoparticles are spherical and have an average particle size of 350-400 nm.

3. The colon-targeted delivery system according to claim 1, wherein: The purity of the amifostine is ≥ 99.0%; The pectin contains ≥ 74.0% galacturonic acid and has a molecular weight of 20,000-40,000 Da; The chitosan has a degree of deacetylation of ≥ 75.0% and a viscosity of 20-300 mPas.

4. The colon targeted delivery system according to claim 1, wherein: 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and triethylamine are also included as cross-linking agents.

5. A method of preparing the colon targeted delivery system according to any one of claims 1 to 4, characterized by: The method comprises the following steps: S1: preparing a nanoparticle core PEC-AMF S1.1: dissolving the pectin in distilled water and stirring to form a solution A; S1.2: adding 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride to the solution A and stirring to form a solution B; S1.3: adding triethylamine and amifostine to the solution B, stirring, dialysis, and freeze-drying to obtain PEC-AMF nanoparticles; S2: preparing CS / PEC-AMF nanoparticles S2.1: weighing the PEC-AMF nanoparticles obtained in S1.3, dissolving them in distilled water, and adjusting the pH of the solution to 5.0 to form a solution C; S2.2: dissolving the chitosan in distilled water and adjusting the pH to 4.5-5.5 to form a solution D; S2.3: adding the solution D to the solution C, stirring, and filtering to obtain CS / PEC-AMF nanoparticles.

6. The method of claim 5, wherein the colon-targeted delivery system is prepared by the steps of: In S1.2, the amount of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride is 1.5-2 times the molar amount of the carboxyl in the pectin; In S1.3, the amount of triethylamine is 1.2-1.5 times the molar amount of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride; The amount of amifostine is 0.5-1 times the molar amount of the carboxyl in the pectin.

7. The method of claim 5, wherein the colon targeting delivery system is prepared by the steps of: In S2.1, the mass ratio of PEC-AMF nanoparticles to distilled water is 1:20-1:50; In S2.2, the concentration of chitosan is 0.5-1.5% w / v; In S2.3, the mass ratio of pectin to chitosan is 2:

1.

8. A method for preparing a colon-targeted delivery system according to claim 5, characterized in that: In S1.1, the stirring rate is 300-600 rpm and the stirring time is 10-25 min; In S1.2, the stirring rate is 300-600 rpm, the stirring time is 10-20 min, and the pH range is 4.5-6.0; In S1.3, the stirring time is 24 hours, the stirring rate is 300-600 rpm, the dialysis conditions are as follows: the molecular weight cut-off value of the dialysis bag is 6,000-8,000 Da, the dialysis time is 48 h, and the dialysis water is changed at least 3 times. In S2.3, the feeding rate is 1-2 mL / min, the stirring rate is 300-500 rpm, and the stirring time is 12-24 h.

9. A method for preparing a colon-targeted delivery system according to claim 5, characterized in that: In S2.3, a polyvinylidene fluoride or nylon filter membrane with a pore size of 0.22 μm is selected.

10. Use of the colon-targeted delivery system according to any one of claims 1-9 in the preparation of a medicament for reducing the degree of radiation-induced intestinal injury.

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