CO Gas Targeted Controlled Release Nanocomposite for the Treatment of Inflammatory Bowel Disease and Its Preparation Method

Abstract

The present invention relates to an oxidative stress-responsive CO gas-controlled release nanocomposite, a preparation method and an application, and provides an efficient, specific and safe oxidative stress-responsive CO gas-controlled release nanocomposite (LBL-CO@MPDA) based on mesoporous polydopamine nanoparticles (MPDA) and a chitosan / sodium alginate polyelectrolyte multilayer film, which effectively solves the oral delivery obstacle of therapeutic CO gas. It can not only target and enrich in the IBD inflammatory microenvironment, but also achieve targeted controlled release of CO therapeutic gas, thereby effectively alleviating oxidative stress, reducing inflammatory responses, restoring immune homeostasis, providing a new option for the treatment of inflammatory bowel disease, solving clinical problems such as limited efficacy, high recurrence rate, drug dependence and large toxic and side effects in the current treatment of IBD, and bringing new hope for the cure of IBD.

Description

Technical Field

[0001] The present invention belongs to a nanocomposite and a delivery system for treating inflammatory bowel disease, and relates to an oxidative stress-responsive CO gas-controlled release nanocomposite, a preparation method thereof, and a CO gas-targeted nanodelivery system for treating inflammatory bowel disease. Background Art

[0002] In recent years, the incidence of inflammatory bowel disease (IBD) has been increasing in China. The precise diagnosis and treatment of IBD have serious problems. The condition of patients often recurs and gradually worsens, and ultimately can lead to the loss of gastrointestinal structure and function, and even canceration, seriously affecting the quality of life of patients and becoming a difficult problem and challenge for social health in China. Currently, the commonly used drugs for treating IBD include 5-aminosalicylic acid preparations, glucocorticoids, immunomodulators, etc. However, the course of IBD is long and long-term medication is required. Although these drugs have certain efficacy in inducing and maintaining clinical remission and promoting mucosal healing, due to systemic administration and non-specific distribution of the drugs, a series of side effects are often inevitably caused.

[0003] Currently, some new biological agents for inflammation regulation, such as anti-TNF-α monoclonal antibodies, provide new strategies and options for the treatment of IBD. However, a considerable number of patients still show primary non-response or secondary loss of response during the treatment process. At the same time, some new adverse reactions and the economic cost of biological agents also limit the clinical application of biological agents. A 10-year clinical evaluation shows that 30-50% of IBD patients do not respond to current anti-inflammatory treatments. Therefore, there is an urgent need to explore safer and more efficient new therapies.

[0004] In recent years, gas therapy, as a new and promising therapeutic strategy, has been developed and applied in the biomedical field, such as the treatment of major diseases such as cardiovascular diseases, cancer, bacterial infections, and neurological diseases. Under physiological conditions, trace amounts of carbon monoxide gas (CO) present in the body play an important regulatory role. In addition, as a high-quality anti-inflammatory gas signaling molecule, CO is not easily consumed under pathological conditions and has unique advantages in inhibiting inflammatory responses. Research has found that CO can not only inhibit inflammation-related signaling pathways such as p38 MAPK, TLR-2, and NF-κB, but also relieve oxidative stress by inducing the activation of heme oxygenase (HO-1), thereby effectively inhibiting the expression of pro-inflammatory factors. Therefore, CO gas therapy can be used as a potential strategy for the treatment of IBD. However, due to the poor water solubility and stability of CO gas prodrugs, there are still great challenges in the precise delivery and on-demand release of CO in the pathological microenvironment. Currently, the nanodelivery strategy that can target and control the release of therapeutic gases is the key to improving the effect of gas therapy!

[0005] Based on this, the present invention aims to provide an efficient, specific, and safe oxidative stress-responsive CO gas-controlled release nanocomposite (LBL-CO@MPDA) based on mesoporous polydopamine nanoparticles (MPDA) and chitosan / sodium alginate polyelectrolyte multilayers, which can effectively solve the oral delivery obstacle of therapeutic CO gas. It can not only target and enrich in the IBD inflammatory microenvironment, but also achieve targeted controlled release of CO therapeutic gas, thereby effectively alleviating oxidative stress, reducing inflammatory responses, and restoring immune homeostasis, providing a new option for the treatment of inflammatory bowel disease and solving current clinical problems such as limited efficacy, high recurrence rate, drug dependence, and large side effects in IBD treatment, bringing new hope for the cure of IBD. Summary of the Invention

[0006] Technical Problems to be Solved

[0007] To avoid the deficiencies of the prior art and explore a new nanomaterial for the treatment of inflammatory bowel disease (IBD), in view of the pathological microenvironment, the present invention proposes an oxidative stress-responsive CO gas-controlled release nanocomposite, a preparation method, and an application thereof. The present invention uses a hydrophobic metal carbonyl complex Fe3(CO) 12 as a CO gas prodrug, which is loaded into the mesoporous structure of mesoporous polydopamine nanoparticles (MPDA) through hydrophobic interaction, and the surface of the nanoparticles is modified with chitosan / sodium alginate (Chi / Alg) polyelectrolyte multilayers by the layer-by-layer self-assembly technique (LBL) to finally form an orally administrable LBL-CO@MPDA nanoparticle, constructing an oxidative stress-responsive CO gas-targeted nanodelivery system (LBL-CO@MPDA) for IBD treatment.

[0008] Technical Solution

[0009] An oxidative stress-responsive CO gas-controlled release nanocomposite, characterized in that: a hydrophobic metal carbonyl complex Fe3(CO) 12 is loaded into the mesoporous structure of mesoporous polydopamine nanoparticles MPDA, and the surface of the nanoparticles is modified with chitosan / sodium alginate Chi / Alg polyelectrolyte multilayers by the layer-by-layer self-assembly technique LBL to form LBL-CO@MPDA nanoparticles.

[0010] A preparation method of the oxidative stress-responsive CO gas-controlled release nanocomposite according to claim 1, characterized by the following steps:

[0011] Step 1: Prepare MPDA nanoparticles by the "template method":

[0012] Step 1a: Dissolve and disperse 0.1 - 0.5 g of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) F127 and 200 - 500 μL of mesitylene TMB in a water / ethanol mixture.

[0013] Step 1b: Add 50 - 100 mg of tris (hydroxymethyl) aminomethane (Tris) to the mixed solution and dissolve it. Then add 50 - 100 mg of dopamine hydrochloride, and react with magnetic stirring at room temperature for 12 - 24 hours.

[0014] Step 1c: Centrifuge to collect the crude product in the reaction solution, and disperse it in an ethanol / acetone mixed solution to remove the organic template.

[0015] Step 1d: Centrifuge to collect the precipitate to obtain MPDA nanoparticles.

[0016] Step 2: Load Fe3(CO) 12 into the mesoporous structure of MPDA to obtain CO@MPDA nanoparticles:

[0017] Step 2a: Disperse the MPDA nanoparticles in chloroform, and then transfer them to a Schlenk reaction flask. Degas the reaction solution by the freeze-thaw pump cycling method and fill it with N2 for protection.

[0018] Step 2b: Dropwise add the chloroform solution dissolved with Fe3(CO) 12 to the MPDA suspension in Step 2a, and react with magnetic stirring in the dark at room temperature for 12 - 48 h.

[0019] Step 2c: Wash the product with ethanol, centrifuge to collect it, and the obtained product is the MPDA nanoparticles loaded with Fe3(CO) 12 which is denoted as CO@MPDA nanoparticles.

[0020] Step 3: Construct a chitosan / sodium alginate (Chi / Alg) polyelectrolyte multilayer film on the surface of CO@MPDA nanoparticles by the layer-by-layer self-assembly technique (LBL) to obtain LBL-CO@MPDA nanoparticles:

[0021] Step 3a: Disperse the CO@MPDA nanoparticles in a chitosan solution with a pH of 5 - 8, react with magnetic stirring at room temperature for 10 - 60 min, centrifuge, and wash with PBS for 1 - 3 times. Collect to obtain Chi-CO@MPDA nanoparticles.

[0022] Step 3b: Suspend the Chi-CO@MPDA nanoparticles in a sodium alginate solution, stir magnetically at room temperature for 10 - 60 min, centrifuge, and wash with PBS for 1 - 3 times. Collect to obtain Chi / Alg-CO@MPDA nanoparticles.

[0023] Repeat Step 3a and Step 3b for 2 - 5 times. Finally, construct a Chi / Alg polyelectrolyte multilayer film on the surface of CO@MPDA to finally obtain LBL-CO@MPDA nanoparticles.

[0024] In step 1a, the water / ethanol mixture contains 50 - 100 mL of deionized water and 50 - 100 mL of ethanol.

[0025] In steps 1b and 2b, the rotation speed of the magnetic stirring reaction is 300 - 500 revolutions per minute.

[0026] In step 1c, the ethanol / acetone mixture contains 15 - 30 mL of ethanol and 5 - 20 mL of acetone.

[0027] In steps 1d, 2c and 3b, the centrifugation parameters are 5000 - 11000 rpm, 4 - 25 °C, and 5 - 30 minutes.

[0028] In steps 3a and 3b, the rotation speed of the magnetic stirring at room temperature is 300 - 500 revolutions per minute.

[0029] In step 3a, the centrifugation parameters are 5000 - 11000 rpm, 4 - 25 °C, and 5 - 30 minutes.

[0030] In step 2b, the concentration of Fe3(CO) 12 is 5 - 30 mg / mL.

[0031] An application of the oxidative stress-responsive CO gas-controlled release nanocomposite, characterized in that: the LBL-CO@MPDA nanoparticles are used for a CO gas-targeted nanodelivery system for the treatment of inflammatory bowel disease (IBD), solving the delivery obstacle of therapeutic CO gas, being able to target and enrich in the IBD inflammatory microenvironment, being able to achieve targeted controlled release of therapeutic CO gas, thereby effectively alleviating oxidative stress, reducing the inflammatory response, and restoring intestinal immune homeostasis.

[0032] Beneficial effects

[0033] An oxidative stress-responsive CO gas-controlled release nanocomposite, preparation method and application provided by the present invention provide an efficient, specific and safe oxidative stress-responsive CO gas-controlled release nanocomposite (LBL-CO@MPDA) based on mesoporous polydopamine nanoparticles (MPDA) and chitosan / sodium alginate polyelectrolyte multilayers, effectively solving the oral delivery obstacle of therapeutic CO gas. It can not only target and enrich in the IBD inflammatory microenvironment, but also achieve targeted controlled release of therapeutic CO gas, thereby effectively alleviating oxidative stress, reducing the inflammatory response, and restoring immune homeostasis, providing a new option for the treatment of inflammatory bowel disease, solving clinical problems such as limited efficacy, high recurrence rate, drug dependence, and large toxic and side effects in the current treatment of IBD, and bringing new hope for the cure of IBD.

[0034] Advantages of the present invention:

[0035] (1) The CO@MPDA nanoparticles can effectively load hydrophobic CO gas prodrugs and achieve responsive delivery of CO gas in the oxidative stress microenvironment of IBD, effectively solving the delivery obstacle of therapeutic CO gas.

[0036] (2) Using the LBL technique, the Chi / Alg polyelectrolyte multilayer film constructed on the surface of CO@MPDA nanoparticles is the key to endowing the nanoparticles with good stability, active targeting, and site-controlled release. On the one hand, the Chi / Alg barrier layer can not only effectively protect the loaded drugs from being damaged by the physical and chemical factors in the gastrointestinal tract and keep the drugs stable before reaching the lesion site, but on the other hand, it can target and enrich to the IBD inflammatory microenvironment through electrostatic interaction, stimulate the degradation of the Chi / Alg "nano-valve", activate the release of CO gas, and thus prolong the action time.

[0037] (3) The preparation method of the present invention does not require special equipment, is simple to operate and has strong controllability, providing a new nano-delivery strategy for the treatment of IBD. Description of the Drawings

[0038] Figure 1 : Transmission electron microscope images of different nanoparticles

[0039] Figure 2 : Changes in the zeta potential of the particle surface during the LBL process

[0040] Figure 3 : Size changes of nanoparticles in simulated gastric juice and simulated intestinal juice (added with 100 μM H2O2)

[0041] Figure 4 : CO release from CO@MPDA nanoparticles after treatment with different concentrations of H2O2

[0042] Figure 5 : Representative fluorescence images of the ROS level in macrophages after different treatments

[0043] Figure 6 : Contents of pro-inflammatory and anti-inflammatory factors secreted by macrophages after different treatments Detailed Embodiments

[0044] The present invention will be further described in combination with the embodiments and the drawings:

[0045] The present invention has developed an oxidative stress-responsive CO gas-controlled release nano-complex and a preparation method, and at the same time, a reactive oxygen species-responsive CO gas-releasing nano-delivery system (LBL-CO@MPDA) using the nano-complex for the treatment of IBD, which is characterized by including the following steps.

[0046] Step 1: Preparation of MPDA nanoparticles

[0047] Prepare MPDA nanoparticles by the "template method": (1) Dissolve and disperse 0.1 - 0.5 g of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (F127) and 200 - 500 μL of mesitylene (TMB) in a water / ethanol mixture (50 - 100 mL of deionized water and 50 - 100 mL of ethanol); (2) Then, add 50 - 100 mg of tris(hydroxymethyl)aminomethane to the above mixture; (3) After complete dissolution, add 50 - 100 mg of dopamine hydrochloride and stir magnetically at a speed of 300 - 500 revolutions per minute at room temperature for 12 - 24 hours; (4) Centrifuge to collect the crude product in the reaction solution and disperse it in an ethanol / acetone mixture (15 - 30 mL of ethanol, 5 - 20 mL of acetone) to remove the organic template; (5) Centrifuge to collect the precipitate, with centrifugation parameters of 5000 - 11000 rpm, 4 - 25 °C, for 5 - 30 minutes. The obtained product is the MPDA nanoparticles.

[0048] Step 2: Preparation of CO@MPDA nanoparticles

[0049] Load Fe3(CO) 12 into the mesoporous structure of MPDA through hydrophobic interaction: (1) Disperse the MPDA nanoparticles obtained in Step 1 in 5 - 30 mL of chloroform, then transfer it to a Schlenk reaction flask, degas the reaction solution by the freeze-thaw pump cycle method, and fill it with N2 for protection; (2) Then, dropwise add a chloroform solution (5 - 30 mg / mL) of dissolved Fe3(CO) 12 to the above MPDA suspension, and stir magnetically at a speed of 300 - 500 revolutions per minute in the dark at room temperature for 12 - 48 h; (3) Subsequently, centrifuge to collect the product, wash it with ethanol, and the centrifugation parameters are 5000 - 11000 rpm, 4 - 25 °C, for 5 - 30 minutes. The obtained product is the MPDA nanoparticles loaded with Fe3(CO) 12 , briefly denoted as CO@MPDA nanoparticles.

[0050] Step 3: Preparation of LBL-CO@MPDA nanoparticles

[0051] Constructing a chitosan / sodium alginate (Chi / Alg) polyelectrolyte multilayer film on the surface of CO@MPDA nanoparticles through the layer-by-layer self-assembly technique (LBL): (1) Disperse the CO@MPDA nanoparticles obtained in step two in a 1-4 mg / mL chitosan solution with a pH of 5-8, and magnetically stir the reaction at a rotation speed of 300-500 revolutions per minute at room temperature for 10-60 minutes. After centrifugation, wash with PBS 1-3 times. The centrifugation parameters are 5000-11000 rpm, 4-25 °C, and 5-30 minutes. Collect the Chi-CO@MPDA nanoparticles; (2) Suspend the obtained nanoparticles in a 1-4 mg / mL sodium alginate solution, and magnetically stir at a rotation speed of 300-500 revolutions per minute at room temperature for 10-60 minutes. After centrifugation, wash with PBS 1-3 times. The centrifugation parameters are 5000-11000 rpm, 4-25 °C, and 5-30 minutes. Collect the Chi / Alg-CO@MPDA nanoparticles; (3) Repeat processes (1) and (2) 2-5 times to finally construct a Chi / Alg polyelectrolyte multilayer film on the surface of CO@MPDA, and finally obtain LBL-CO@MPDA nanoparticles.

[0052] Combined with the design concept of LBL-CO@MPDA nanoparticles, we believe that:

[0053] First, patients with inflammatory bowel disease orally take LBL-CO@MPDA nanoparticles. The nanoparticles enter the stomach, and the LBL barrier layer modified on their surface can isolate the encapsulated molecules from contact with gastric acid and digestive enzymes in the gastrointestinal tract, effectively protecting the CO@MPDA nanoparticles from being damaged by the harsh physiological environment of the gastrointestinal tract, so that the drug is not released in the stomach, duodenum, jejunum, and the front end of the ileum after oral administration, realizing the high stability of the nanoparticles in the gastrointestinal tract.

[0054] Then, due to the presence of high concentrations of positively charged proteins (eosinophil cationic protein and transferrin) in the intestinal inflammatory tissue, the surface of the intestinal mucosal epithelial cells at the inflammatory site is positively charged, while the surface of the LBL-CO@MPDA nanoparticles we invented is strongly negatively charged. When the nanoparticles reach the intestinal inflammatory site, the electrostatic interaction causes the nanoparticles to target and adhere to the inflammatory tissue and accumulate locally at the lesion.

[0055] Finally, at the intestinal inflammatory site, there is a local high concentration of oxidative stress signal, which stimulates the degradation of the Chi / Alg "nano-valve", activates the targeted controlled release of CO gas, gives full play to its gas therapy effect, thereby effectively relieving oxidative stress, reducing the inflammatory response, restoring immune homeostasis, and ultimately promoting intestinal mucosal repair.

[0056] Specific embodiments relate to the preparation of an oxidative stress-responsive CO gas-controlled release nanocomposite, including the amounts of F127 and TMB, the rotation speed of the stirring reaction, the ratio of the ethanol / acetone mixture, the concentration of Fe3(CO) 12 12, the reaction time, the concentrations of chitosan and sodium alginate, and the selection of the number of layer-by-layer self-assembly repetitions:

[0057] Example 1:

[0058] Step 1: Preparation of MPDA nanoparticles

[0059] MPDA nanoparticles were prepared by the "template method": (1) 0.36 g of poly(ethylene glycol)-block-poly(propylene glycol) (F127) and 450 μL of mesitylene (TMB) were dissolved and dispersed in a water / ethanol (65 mL of deionized water, 60 mL of ethanol) mixture; (2) then, 90 mg of tris(hydroxymethyl)aminomethane was added to the above mixture; (3) after complete dissolution, 60 mg of dopamine hydrochloride was added, and the reaction was magnetically stirred at a rotation speed of 300 rpm at room temperature for 24 hours; (4) the crude product in the reaction solution was collected by centrifugation and dispersed in an ethanol / acetone mixture (20 mL of ethanol, 10 mL of acetone) to remove the organic template; (5) the precipitate was collected by centrifugation, and the centrifugation parameters were 11000 rpm, 4 °C, 15 minutes. The obtained product was MPDA nanoparticles.

[0060] Step 2: Preparation of CO@MPDA nanoparticles

[0061] Fe3(CO)12 was loaded into the mesoporous structure of MPDA through hydrophobic interaction: (1) the obtained MPDA nanoparticles were dispersed in 10 mL of chloroform, then transferred to a Schlenk reaction flask, and the reaction solution was degassed by the freeze-thaw pump cycle method and protected with N2; (2) then, a chloroform solution (10 mg / mL) of Fe3(CO)12 was added dropwise to the above MPDA suspension, and the reaction was magnetically stirred at a rotation speed of 500 rpm at room temperature in the dark for 48 h; (3) subsequently, the product was collected by centrifugation, washed with ethanol, and the centrifugation parameters were 11000 rpm, 4 °C, 15 minutes. The obtained product was MPDA nanoparticles loaded with Fe3(CO)12, abbreviated as CO@MPDA nanoparticles.

[0062] Step 3: Preparation of LBL-CO@MPDA nanoparticles

[0063] Construct a chitosan / sodium alginate (Chi / Alg) polyelectrolyte multilayer film on the surface of CO@MPDA nanoparticles through the layer-by-layer self-assembly technique (LBL): (1) Disperse CO@MPDA nanoparticles in a 2 mg / mL chitosan solution with a pH of 6, magnetically stir the reaction at a speed of 500 revolutions per minute at room temperature for 30 min, wash 3 times with PBS after centrifugation, and the centrifugation parameters are 11,000 rpm, 4 °C, and 15 minutes. Collect the Chi-CO@MPDA nanoparticles; (2) Suspend the obtained nanoparticles in a 2 mg / mL sodium alginate solution, magnetically stir the reaction at a speed of 500 revolutions per minute at room temperature for 30 min, wash 3 times with PBS after centrifugation, and the centrifugation parameters are 11,000 rpm, 4 °C, and 15 minutes. Collect the Chi / Alg-CO@MPDA nanoparticles; (3) Repeat processes (1) and (2) a total of 3 times for this process. Finally, construct a Chi / Alg polyelectrolyte multilayer film on the surface of CO@MPDA to finally obtain LBL-CO@MPDA nanoparticles.

[0064] The product obtained by the above preparation method is an active oxygen-responsive CO gas-releasing nanodelivery system for the treatment of IBD. During the construction process, many factors will affect the synthesis of MPDA, CO@MPDA, and LBL-CO@MPDA nanoparticles and the release of responsive CO gas. For example, the amounts of F127 and TMB in the MPDA synthesis process, the rotation speed of the stirring reaction, the ratio of the ethanol / acetone mixture, the concentration of Fe3(CO)12, the reaction time, the concentrations of chitosan and sodium alginate, the number of layer-by-layer self-assembly repetitions, etc. Different control conditions will directly affect the construction of functional nanoparticles.

[0065] Example 2: Change the amounts of F127 and TMB in the MPDA synthesis process, the rotation speed of the stirring reaction, the ratio of the ethanol / acetone mixture, Fe3(CO) 12 The concentration, reaction time, concentrations of chitosan and sodium alginate, and the number of layer-by-layer self-assembly repetitions are as follows: Use 0.36 g of F127 and 450 μL of TMB as the organic template, the rotation speed of the stirring reaction is 300 revolutions per minute, and use the ethanol / acetone mixture (20 mL of ethanol, 10 mL of acetone) to remove the organic template to obtain MPDA nanoparticles with a uniform morphology and a good mesoporous structure.

[0066] Example 3: Change the amounts of F127 and TMB in the MPDA synthesis process, the rotation speed of the stirring reaction, the ratio of the ethanol / acetone mixture, Fe3(CO) 12 The concentration, reaction time, concentrations of chitosan and sodium alginate, and the number of layer-by-layer self-assembly repetitions are as follows: Use 10 mg / mL of Fe3(CO) 12Reacting with the obtained MPDA in the dark for 48 h can effectively achieve the loading of the hydrophobic CO gas prodrug; when the concentrations of chitosan and sodium alginate are both 2 mg / mL and the LBL is repeated 3 times, the obtained LBL-CO@MPDA nanoparticles have a strong negative charge on the surface, which can effectively resist the destruction of gastric juice and achieve the CO gas-responsive delivery mediated by the oxidative stress microenvironment.

[0067] As can be seen from the attached drawings obtained in the above embodiments:

[0068] Figure 1 The transmission electron microscope images of MPDA, CO@MPDA, and LBL-CO@MPDA nanoparticles Figure 1 show that the obtained MPDA nanoparticles have a diameter of about 200 nm and have a good mesoporous structure, which is beneficial to the loading of Fe3(CO) 12 ; the mesoporous structure of the obtained CO@MPDA nanoparticles disappears, and at the same time, a uniform distribution of Fe elements is observed in the EDS spectral image of the CO@MPDA nanoparticles, indicating the successful loading of Fe3(CO) 12 ; the obtained LBL-CO@MPDA nanoparticles have a diameter distribution of about 300 nm, and a multi-layer film structure is clearly visible on the surface, and the thickness of the Chi / Alg multi-layer film is about 50 nm;

[0069] Figure 2 It is the change of the surface zeta potential during the construction of LBL-CO@MPDA nanoparticles, Figure 2 showing that after the polycationic Chi is coated, the zeta potential of CO@MPDA changes from -38.2 mV to 4.4 mV of Chi-CO@MPDA, and then after the polyanionic Alg is coated, the zeta potential drops to -34.3 mV of Chi / Alg-CO@MPDA; similar potential changes are also observed during the further Chi / Alg coating treatment; after 3 cycles of Chi / Alg coating, the zeta potential of LBL-CO@MPDA finally maintains at -45.6 mV; therefore, the obtained LBL-CO@MPDA nanoparticles have a strong negative charge on the surface, which will be beneficial to enriching to the positively charged inflammatory bowel mucosa site through electrostatic interaction;

[0070] Figure 3 It is the size change of LBL-CO@MPDA nanoparticles after being treated in simulated gastric juice and simulated intestinal fluid (added with 100 μM of H2O2) for different times, Figure 3It was shown that after incubation in simulated gastric juice for 4 h, the size of the nanoparticles did not decrease, indicating that the LBL shell remained relatively intact; while in simulated intestinal fluid (added with 100 μM H2O2), the size of the nanoparticles decreased from 313.4 nm to 279.7 nm, indicating that the Chi / Alg coating was degraded; therefore, the Chi / Alg multilayer film on the surface of the obtained LBL-CO@MPDA nanoparticles could effectively resist the damage of gastric juice and achieve the delivery of CO@MPDA nanoparticles at the site of inflammatory bowel disease;

[0071] Figure 4 For the release of CO gas from CO@MPDA nanoparticles under in vitro oxidative stress conditions (different concentrations of H2O2), Figure 4 it was shown that after incubation for 60 min, the release of CO showed a H2O2 concentration dependence. When the concentrations were 5, 50, and 100 μM, the release amounts of CO reached 0.84, 2.92, and 3.59 μM, respectively; therefore, the obtained CO@MPDA nanoparticles could achieve oxidative stress microenvironment-mediated CO gas-responsive delivery.

[0072] From Figures 1-4 it can be seen that the LBL-CO@MPDA nanoparticles prepared in the present invention have good oral bioavailability and the potential to achieve reactive oxygen species-responsive CO gas delivery on demand at the site of inflammatory bowel disease.

[0073] The LBL-CO@MPDA nanoparticles prepared in the present invention are used for a CO gas-targeted nanodelivery system for the treatment of inflammatory bowel disease. An example for evaluating the anti-inflammatory effect of the constructed CO@MPDA nanoparticles in vitro is as follows:

[0074] In this example, normal macrophages (RAW264.7, 5×10 4 cells / cm 2 ) were used as model cells and pretreated with H2O2 solution (100 μM) for 12 h, and then the activated macrophages were co-incubated with MPDA and CO@MPDA nanoparticles (100 μg / mL) to explore the ROS scavenging ability and anti-inflammatory effect of CO@MPDA nanoparticles. The experimental groups included a blank control group (normal), an H2O2 treatment group (H2O2), an H2O2 + MPDA group, and an H2O2 + CO@MPDA group. The specific experiments included:

[0075] Experiment 1:

[0076] After co-culture for 24 h and 48 h, 500 μL of the ROS fluorescent probe 2’,7’–dichlorofluorescein diacetate (DCFH-DA, 10 μM) was added to each group;

[0077] After incubation at 37°C for 30 minutes, gently rinse 3 times with PBS buffer;

[0078] Use an inverted fluorescence microscope to observe the ROS level inside the cells.

[0079] Experiment 2:

[0080] After co-culturing for 48 h, collect the culture media of each group, and use an ELISA kit to detect the secretion levels of pro-inflammatory factors (TNF-α, IL-6) and anti-inflammatory factor (IL-10) regulated by different nanoparticles in macrophages, respectively.

[0081] In this example:

[0082] Figure 5 are representative fluorescence images of the ROS level in macrophages after different treatments. The stronger the green fluorescence, the higher the ROS level. Figure 5 It shows that macrophages stimulated by H2O2 exhibit obvious green fluorescence, while after treatment with MPDA, the green fluorescence in macrophages is significantly weakened. In contrast, the green fluorescence in the CO@MPDA treatment group is the weakest, further reducing the level of oxidative stress; Therefore, the CO@MPDA nanoparticles effectively reduce the oxidative stress damage caused by H2O2 to macrophages;

[0083] Figure 6 are the contents of pro-inflammatory and anti-inflammatory factors secreted by macrophages after different treatments. Figure 6 It shows that the stimulation of H2O2 exacerbates the secretion of pro-inflammatory factors (TNF-α and IL-6) in macrophages. After MPDA treatment, the content of TNF-α secreted by cells is significantly reduced. Compared with MPDA, the CO@MPDA treatment significantly inhibits the secretion of pro-inflammatory factors TNF-α and IL-6 while significantly promoting the secretion of anti-inflammatory factor IL-10; Therefore, CO@MPDA has good anti-inflammatory activity.

[0084] In summary, the preparation method in the present invention does not require special equipment, is simple to operate and has strong controllability. It successfully constructs a reactive oxygen species-responsive CO gas-targeted nanodelivery system that can be used for the treatment of inflammatory bowel disease, effectively solves the delivery obstacle of therapeutic CO gas, and it can not only target and enrich in the IBD inflammatory microenvironment, but also achieve targeted controlled release of CO therapeutic gas, thereby effectively alleviating oxidative stress and reducing the inflammatory response, providing a new strategy for the treatment of inflammatory bowel disease, and to a certain extent solving the problems of limited curative effect, high recurrence rate, drug dependence, and large toxic and side effects in the current clinical treatment of IBD.

Claims

1. An oxidation stress-responsive CO gas-controlled release nanocomposite, characterized in that: Loading the hydrophobic metal carbonyl complex Fe3(CO) 12 into the mesoporous structure of mesoporous polydopamine nanoparticles MPDA, and using the layer-by-layer self-assembly technique LBL to modify the surface of the nanoparticles with a chitosan / sodium alginate Chi / Alg polyelectrolyte multilayer film to form LBL-CO@MPDA nanoparticles.

2. The preparation method of the oxidation stress-responsive CO gas-controlled release nanocomposite according to claim 1, characterized in that The steps are as follows: Step 1: Prepare MPDA nanoparticles by the "template method": Step 1a: Dissolve and disperse 0.1 - 0.5 g of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) F127 and 200 - 500 μL of mesitylene TMB in a water / ethanol mixture; Step 1b: After adding 50 - 100 mg of tris(hydroxymethyl)aminomethane Tris to the mixture and dissolving it, add 50 - 100 mg of dopamine hydrochloride and react with magnetic stirring at room temperature for 12 - 24 hours; Step 1c: Centrifuge to collect the crude product in the reaction solution and disperse it in an ethanol / acetone mixture to remove the organic template; Step 1d: Centrifuge to collect the precipitate to obtain MPDA nanoparticles; Step 2: Load Fe3(CO) 12 into the mesoporous structure of MPDA to obtain CO@MPDA nanoparticles: Step 2a: Disperse the MPDA nanoparticles in chloroform, then transfer them to a Schlenk reaction flask, degas the reaction solution by the freeze-thaw pump cycle method, and fill with N2 for protection; Step 2b: Slowly add dropwise the chloroform solution dissolving Fe3(CO) 12 to the MPDA suspension in Step 2a, and magnetically stir the reaction for 12 - 48 h at room temperature in the dark; Step 2c: After washing the product with ethanol, centrifuge and collect it. The resulting product is the MPDA nanoparticles loaded with Fe3(CO) 12 , denoted as CO@MPDA nanoparticles; Step 3: Construct a chitosan / sodium alginate Chi / Alg polyelectrolyte multilayer film on the surface of CO@MPDA nanoparticles by the layer-by-layer self-assembly technique LBL to obtain LBL-CO@MPDA nanoparticles: Step 3a: Disperse the CO@MPDA nanoparticles in a chitosan solution with a pH of 5 - 8, react with magnetic stirring at room temperature for 10 - 60 min, centrifuge and wash with PBS 1 - 3 times, and collect to obtain Chi-CO@MPDA nanoparticles; Step 3b: Suspend the Chi-CO@MPDA nanoparticles in a sodium alginate solution, stir magnetically at room temperature for 10 - 60 min, centrifuge and wash with PBS 1 - 3 times, and collect to obtain Chi / Alg-CO@MPDA nanoparticles; Repeat Step 3a and Step 3b a total of 2 - 5 times, finally construct a Chi / Alg polyelectrolyte multilayer film on the surface of CO@MPDA, and finally obtain LBL-CO@MPDA nanoparticles.

3. The method according to claim 2, wherein: In Step 1a, the water / ethanol mixture is 50 - 100 mL of deionized water and 50 - 100 mL of ethanol.

4. The method according to claim 2, wherein: In Step 1b and Step 2b, the rotation speed of the magnetic stirring reaction is 300 - 500 revolutions per minute.

5. The method according to claim 2, wherein: In Step 1c, the ethanol / acetone mixture is 15 - 30 mL of ethanol and 5 - 20 mL of acetone.

6. The method according to claim 2, wherein: The centrifugation parameters in Step 1d, Step 2c and Step 3b are 5000 - 11000 rpm, 4 - 25 °C, 5 - 30 minutes.

7. The method according to claim 2, wherein: In Step 3a and Step 3b, the rotation speed of the magnetic stirring at room temperature is 300 - 500 revolutions per minute.

8. The method according to claim 2, wherein: The centrifugation parameters in Step 3a are 5000 - 11000 rpm, 4 - 25 °C, 5 - 30 minutes.

9. The method according to claim 2, characterized in that: The concentration of Fe3(CO) in step 2b 12 is 5 to 30 mg / mL.

Images