Prebiotic hydrogel coated nano motor sustained release preparation and preparation method thereof

Abstract

The invention relates to the technical field of medicine preparation, and particularly discloses a prebiotic hydrogel coated nano-motor sustained release preparation and a preparation method thereof.The prebiotic hydrogel coated nano-motor sustained release preparation comprises a nano-motor which comprises mesoporous silica nano-particles and amino groups modified on the surfaces of the mesoporous silica nano-particles, the manganese oxide layer is asymmetrically coated on the surface of the mesoporous silicon dioxide nano particle; the prebiotic hydrogel is used for wrapping the nano motor; the at least one active ingredient is loaded in the nanomotor and / or the prebiotic hydrogel and is used for treating inflammation or regulating intestinal flora; based on the active driving characteristic of a nano motor, hydrogen peroxide at an inflammation part is used as fuel, oxygen is generated through catalysis, a drug carrying system is pushed to break through a colon mucus barrier, inulin hydrogel provides a gastric acid protection barrier, and it is ensured that the drug is completely delivered to the colon.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug preparation, and particularly relates to a prebiotic hydrogel-coated nanomotor sustained-release preparation and a preparation method thereof. Background Art

[0002] Ulcerative colitis (UC) is a chronic inflammatory bowel disease, characterized by recurrent colon inflammation, intestinal flora imbalance, and a vicious cycle of inflammation and flora imbalance. In this process, intestinal inflammation causes great damage to the intestinal flora, resulting in an imbalance of the normal flora structure, a sharp reduction in the number of beneficial flora, and a large proliferation of harmful flora. And this flora imbalance in turn further aggravates intestinal inflammation. The two interact with each other to form a vicious cycle, making the condition increasingly difficult to control. At the same time, macrophages will be attracted to the inflammatory area and polarized into M1 type locally, further promoting the exacerbation of inflammation. The existing treatment methods mainly rely on oral anti-inflammatory drugs.

[0003] However, when actually used to treat UC, the efficacy of oral drugs faces many intractable problems. On the one hand, in the inflamed colon, oral drugs have the problems of inaccurate targeting and short retention time, and it is difficult to continuously and stably act on the inflammatory site. On the other hand, oral drugs encounter obstacles when penetrating the mucus barrier and cannot fully reach the lesion area to exert their efficacy. Particularly critically, excessive inflammatory cytokines are generated in the colon, and it is difficult to effectively inhibit these cytokines simply by relying on the dose of oral drugs, which greatly restricts the therapeutic effect of oral drugs. Summary of the Invention

[0004] The purpose of the present invention is to provide a prebiotic hydrogel-coated nanomotor sustained-release preparation and a preparation method thereof to solve the problems raised in the above background art.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] A prebiotic hydrogel-coated nanomotor sustained-release preparation, comprising:

[0007] A nanomotor, which comprises mesoporous silica nanoparticles, amino groups modified on the surface of the mesoporous silica nanoparticles, and a manganese oxide layer asymmetrically coated on the surface of the mesoporous silica nanoparticles;

[0008] A prebiotic hydrogel, which wraps the nanomotor;

[0009] At least one active ingredient, which is loaded in the nanomotor and / or the prebiotic hydrogel and is used for treating inflammation or regulating intestinal flora.

[0010] Preferably, the prebiotic hydrogel is one or a combination of inulin, fructooligosaccharide, and galactooligosaccharide.

[0011] Preferably, the active ingredient includes a traditional Chinese medicine monomer, an anti-inflammatory drug, an antioxidant, or a combination thereof.

[0012] Preferably, the traditional Chinese medicine monomer is glycyrrhizic acid or a derivative thereof.

[0013] Preferably, the manganese oxide layer is formed by an oxidation etching reaction and is used to generate gas in response to reactive oxygen species to drive the movement of the nanomotor.

[0014] A method for preparing the sustained-release preparation according to any one of the above, comprising the following steps:

[0015] S1. Prepare amino-functionalized mesoporous silica nanoparticles;

[0016] S2. Form a manganese oxide layer on the surface of the amino-functionalized mesoporous silica nanoparticles to obtain a nanomotor;

[0017] S3. Mix the nanomotor with the active ingredient so that the active ingredient is loaded into the nanomotor;

[0018] S4. Combine the nanomotor loaded with the active ingredient with a prebiotic hydrogel to form a sustained-release preparation encapsulating the nanomotor.

[0019] Preferably, in step S2, the manganese oxide layer is formed by one of the Pickering emulsion method and the oxidation etching method.

[0020] Preferably, in step S4, the prebiotic hydrogel is dissolved by heating and then mixed with the nanomotor, and a gel structure is formed after cooling or standing.

[0021] Use of the sustained-release preparation according to any one of the above in the preparation of a drug for treating inflammatory bowel disease, regulating the intestinal flora, or scavenging reactive oxygen species.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] (1) Based on the active driving characteristics of the nanomotor, using hydrogen peroxide at the inflammatory site as fuel, catalyzing the generation of oxygen, and promoting the drug delivery system to break through the colonic mucus barrier. The inulin hydrogel provides a gastric acid protection barrier to ensure the complete delivery of the drug to the colon.

[0024] (2) Glycyrrhizic acid directly inhibits the secretion of inflammatory factors, and the manganese dioxide nanomotor significantly reduces the level of reactive oxygen species in the inflammatory region by catalyzing the decomposition of hydrogen peroxide.

[0025] (3) The inulin hydrogel, as a prebiotic, selectively promotes the proliferation of beneficial bacteria such as Bifidobacterium and Lactobacillus, and restores the integrity of the intestinal barrier. Description of the Drawings

[0026] Figure 1 Transmission electron microscopy image of inulin gel of the nano - motor loaded with the traditional Chinese medicine monomer glycyrrhizic acid system of the present invention;

[0027] Figure 2 Scanning electron microscopy images of inulin hydrogel and inulin hydrogel loaded with nano - motors of the present invention;

[0028] Figure 3 Infrared spectrum diagram of the nano - motor modified with silica and manganese dioxide of the present invention;

[0029] Figure 4 Experimental diagram of injecting the inulin gel of the nano - motor loaded with the traditional Chinese medicine monomer glycyrrhizic acid system into water using syringes of different sizes of the present invention;

[0030] Figure 5 Movement trajectories of the nano - motors of the present invention under different hydrogen peroxide concentration conditions;

[0031] Figure 6 Variation diagram of motor performance parameters of the nano - motors of the present invention under different hydrogen peroxide concentrations;

[0032] Figure 7 Absorbance concentration standard curve of glycyrrhizic acid in Example 3 of the present invention;

[0033] Figure 8 Drug - loading rate time curve of glycyrrhizic acid by nano - motors in Example 3 of the present invention;

[0034] Figure 9 CCK8 detection bar chart of cell viability change over time of Raw264.7 cells under different treatment conditions in Example 3 of the present invention;

[0035] Figure 10 CCK8 detection bar chart of cell viability change over time of Caco - 2 cells under different treatment conditions in Example 3 of the present invention;

[0036] Figure 11 Cell live - dead staining fluorescence image under different treatment conditions in Example 3 of the present invention;

[0037] Figure 12 Flow cytometry analysis diagram of intracellular ROS levels under different treatment conditions in Example 4 of the present invention;

[0038] Figure 13 DAI score change diagram under different treatment conditions in Example 5 of the present invention;

[0039] Figure 14 Body weight change diagram under different treatment conditions in Example 5 of the present invention;

[0040] Figure 15 It is a bar graph showing the influence of different treatment groups on the intestinal length in Example 5 of the present invention;

[0041] Figure 16 It is a HE staining section diagram of the colon tissue of mice in different treatment groups in Example 5 of the present invention;

[0042] Figure 17 It is a bar graph showing the influence of different treatment groups on the mRNA expression of TNF-α, il-6, and il-β in Example 5 of the present invention;

[0043] Figure 18 It is a Western Blot diagram showing the influence of different treatment groups on the expression of multiple proteins in Example 5 of the present invention;

[0044] Figure 19 It is a stacked bar graph of the relative abundances of microbial communities in different treatment groups in Example 5 of the present invention. Detailed implementation manners

[0045] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0046] Example 1: Preparation of a prebiotic hydrogel-coated nanomotor sustained-release preparation

[0047] S1. Preparation of amino-functionalized mesoporous silica nanoparticles (NH2-MSNs):

[0048] Add 0.5 g of cetyltrimethylammonium bromide (CTAB), 10 mL of absolute ethanol, 70 mL of ultrapure water, and 0.8 mL of 25% ammonia water into a 100 mL three-necked flask, ultrasonically disperse for 10 min to form a homogeneous micelle system, slowly add 2.5 mL of tetraethyl orthosilicate (TEOS), magnetically stir at 60 °C and 1000 r / min for 3 h to generate a mesoporous silica framework, add 250 μL of 3-aminopropyltriethoxysilane (APTES), raise the temperature to 70 °C, and continue stirring for 3 h to complete the amino-functionalization modification. The product is centrifugally washed with ethanol (10000 r / min, 10 min, repeated 3 times), and white powder of NH2-MSNs is obtained after freeze-drying:

[0049] S2. Preparation of manganese oxide-modified nanomotors:

[0050] Mix 8 g of paraffin wax, 250 mg of NH2-MSNs, and 100 mL of ultrapure water, stir at 70 °C and 1000 rpm for 3 h, wash the product by centrifugation with ethanol (10000 r / min, 10 min, repeat 3 times), and perform freeze-drying treatment on the product to form Wax@NH2-MSNs composite. Mix 1 g of Wax@NH2-MSNs with 200 mg of potassium permanganate dissolved in 10 mL of ultrapure water, stir magnetically at room temperature (25 °C) (600 r / min) for 12 - 24 h, and generate MnO2-modified nanomotors (Motor) through oxidative etching reaction;

[0051] S3. Loading of glycyrrhizic acid and composite of hydrogel:

[0052] Weigh the nanomotor (Motor) and glycyrrhizic acid (GA) according to the mass ratio of 1:2, add 10 mL of absolute ethanol, stir at 800 r / min for 8 h, wash the product by centrifugation with ultrapure water (10000 r / min, 10 min, repeat 3 times), and then perform freeze-drying to obtain Motor@GA. Dissolve 12 mg of Motor@GA and 3.6 g of inulin in 6 mL of ultrapure water, stir at 80 °C for 6 h, and let it stand overnight at room temperature to form a sustained-release preparation encapsulated by inulin hydrogel.

[0053] Example 2: Morphology characterization

[0054] It was observed by transmission electron microscopy (TEM) that the nanomotors were spherical and the surface was uniformly coated with an MnO2 layer ( Figure 1 ); it was observed by scanning electron microscopy (SEM) that the inulin hydrogel presented a three-dimensional network structure and the nanomotors were uniformly dispersed therein ( Figure 2 );

[0055] Weigh 1 - 2 mg of the nanomotor sample and 100 - 200 mg of dry potassium bromide in an agate mortar, grind for 10 - 15 minutes under an infrared lamp to prevent moisture absorption, then transfer to a mold, press into tablets under a pressure of 10 - 15 MPa for 3 - 5 minutes, turn on the Fourier transform infrared spectrometer, preheat for 30 - 60 minutes, set the scanning parameters, the scanning range is 400 - 4000 cm -1 , the number of scans is 32 - 64 times, the resolution is 4 cm -1 , put the prepared sample into the sample cell of the spectrometer, close the cell cover, first perform a background scan to eliminate impurity interference, and then scan the sample to collect infrared absorption signals. It was observed by Fourier transform infrared spectroscopy (FTIR) that NH2-MSNs showed an amino characteristic peak at 500 - 700 cm -1 , and a vibration peak of the Mn - O bond appeared after MnO2 modification ( Figure 3 );

[0056] The inulin gel loaded with the nanomotor-loaded Chinese medicine monomer glycyrrhizic acid system obtained in this example was loaded into syringes of different sizes and then injected into water to verify its injectability ( Figure 4 ).

[0057] Example 3: Motion recording and analysis of nanomotors

[0058] The motion behavior of the nanomotor was recorded using the Malvern Nanoparticle Tracking Analyzer NS500 for 10 seconds, and the motion trajectory of the nanomotor under different hydrogen peroxide concentrations (0mM, 0.1mM, 1mM, 5mM, 10mM) was recorded. Subsequently, referring to the previous research report, the nanomotor tracking image sequence and speed were manually analyzed using the ImageJ plug-in, and the formula MSD(Δt)=<xi[t+Δt)-xi(t)]2> (i = 2 for two-dimensional analysis) to determine the average mean square displacement value (e.g. Figures 5 - 6 ).

[0059] Example 3: Drug performance test

[0060] By preparing 5, 10, 20, 30, and 40 μg / mL glycyrrhizic acid standard solutions and measuring their ultraviolet absorption values, a standard curve of concentration-absorption value was obtained. The ultraviolet absorption peak of glycyrrhizic acid was at 290 nm, and the linear equation was y=8.6858x-0.0295 (R2=0.9976) ( Figure 7 );

[0061] Accurately weigh 5 mg of glycyrrhizic acid and dissolve it in 10 mL of anhydrous ethanol. Add 2.5 mg of nanomotors and disperse it ultrasonically. Place the dispersion on a magnetic stirrer and stir for 2 h, 4 h, 6 h and 8 h, respectively. At each predetermined time point, centrifuge the mixed stock solution at 10,000 r / min for 10 min, aspirate the supernatant, and use an ultraviolet spectrophotometer to determine the glycyrrhizic acid content in the supernatant at each time point. The glycyrrhizic acid content in the supernatant at each time point is calculated by the formula [(initial total amount of glycyrrhizic acid - amount of glycyrrhizic acid in the supernatant at each time point) ÷ mass of the nanomotor-loaded glycyrrhizic acid system] × 100%. Obtain the drug loading rate data for different stirring times to analyze the drug loading process. After stirring for 8 h, the drug loading rate reached 78.4%, indicating that the nanomotor is highly efficient in loading glycyrrhizic acid ( Figure 8 ).

[0062] Example 3: Biosafety Verification

[0063] Use serum-free DEME high-glucose medium to prepare the following different groups of materials:

[0064] Control: fresh culture medium;

[0065] Glycyrrhizic acid group (GA): Glycyrrhizic acid was added to the culture medium at a concentration of 50 μg mL -1 ;

[0066] Nanomotor group (MOTOR): Glycyrrhizic acid was added to the culture medium at a concentration of 50 μg·mL -1 ;

[0067] Glycyrrhizic acid-loaded nanomotor group (GA@MOTOR): Glycyrrhizic acid was added to the culture medium at a concentration of 50 μg·mL -1 ;

[0068] CCK8 assay: Raw264.7 and Caco-2 cells were seeded in 96-well plates at a density of 1×10 4 cells per well and cultured overnight. After the cells adhered and grew normally, each group of materials was added. 10 μL of CCK-8 was added to the culture system, and incubation continued for 2 hours. After incubation, the OD value was measured at a reference wavelength of 450 nm using a multimode plate reader. The cell viability was calculated according to the formula [A (experimental well) - A (blank well)] / [A (control well) - A (blank well)] × 100%, and the effects of related substances and transport systems on cells were evaluated. The cell viability was > 90% ( Figures 9 - 10 );

[0069] Live and dead cell staining: Prepare a kit containing a live cell fluorescent dye (such as Calcein-AM) and a dead cell fluorescent dye (such as PI). Take out the 96-well plate from the incubator, aspirate the culture medium, wash the cells 3 times with PBS buffer for 3 minutes each time to remove residual culture medium and impurities. Dilute Calcein-AM and PI to appropriate concentrations with PBS buffer (Calcein-AM is 1 - 5 μM, PI is 5 - 20 μM), and mix them in equal volumes to make a mixed staining solution. Add 100 - 150 μL of the mixed staining solution to each well to immerse the cells. Incubate in a 37°C, 5% CO2 incubator for 15 - 30 minutes, wash the cells 3 times with PBS buffer to remove unbound dyes, and observe under a fluorescence microscope. Live cells show green fluorescence, and dead cells show blue fluorescence. The cell survival status was evaluated by counting the number of fluorescent cells of different colors. The proportion of live cells (green fluorescence) was > 95%, and the number of dead cells (blue fluorescence) was extremely low ( Figure 11 );

[0070] Example 4: Reactive oxygen species scavenging ability

[0071] Five groups of materials, namely Control, 1 mM H2O2, 1 mM H2O2 + 50 μg / mL GA, 1 mM H2O2 + 50 μg / mL MOTOR, and 1 mM H2O2 + 50 μg / mL GA@MOTOR, were prepared with serum-free DEME high-glucose medium. The density was 6×10 5A Raw264.7 cell suspension at a density of 6 cells / mL was seeded in a 6-well plate. After the cells adhered, in the experimental group, they were incubated with 10 μM hydrogen peroxide solution and each material for 12 hours, while the blank control group was cultured with conventional medium. After the incubation, the supernatant was discarded, and the cells were rinsed 2 - 3 times with PBS. Then, 10 μM DCFH-DA staining reagent was added, and the cells were stained at 37 °C for 30 minutes. The cells were collected using a cell scraper or pipette, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended with PBS. After counting, Figure 12 cells were pipetted into a new centrifuge tube, resuspended with 200 - 400 μL PBS again, and the cell suspension was loaded onto the machine. Fluorescence signal data was detected using a flow cytometer, and the data was analyzed with FlowJo software to draw conclusions. Flow cytometry showed that the intracellular reactive oxygen species (ROS) level in the GA@Motor group was reduced by 65% compared with the H2O2 group (

[0072] Example 5: Therapeutic effect on colitis

[0073] Normal mice were randomly divided into 6 groups (8 mice in each group), namely Control group (PBS + normal water), DSS group (PBS + 4% DSS), In group (inulin gel + 4% DSS), GA group (glycyrrhizic acid + 4% DSS), MOTOR group (inulin gel containing nanomotors + 4% DSS), and IMG group (inulin gel loaded with a nanomotor-loaded glycyrrhizic acid system + 4% DSS). The mice in the experimental groups were first given water containing 4% DSS for 7 days to induce a disease model, and then changed to normal drinking water. The mice in the dosing groups were gavaged from day 1 to day 7. The body weight, fecal consistency, and bleeding of the mice were observed and recorded daily, and the disease activity index (DAI) was calculated to evaluate the disease status.

[0074] The disease activity index (DAI) was obtained by adding the fecal consistency index (ranging from 0 - 3), fecal bleeding index (ranging from 0 - 3), and body weight loss index (ranging from 0 - 4). The disease status of the mice was evaluated through this comprehensive index. The DAI score of the IMG group (drug-loaded gel) was reduced by 70% compared with the DSS group ( Figures 13 - 14 )

[0075] On the 9th day, the mice were euthanized, the colon length was measured, and 1 cm of distal tissue was taken for HE staining to observe the tissue morphology. HE staining showed that the integrity of the colon mucosa in the IMG group was significantly restored, and the infiltration of inflammatory cells was reduced ( Figures 15 - 16 )

[0076] The remaining colon tissues were homogenized to extract proteins and RNA. Western blot was used to detect the protein expression of ZO-1 and OC-1, and q-pcr was used to detect the gene expression of TNF-α, il-6, and il-β ( Figures 17 - 18 )

[0077] Fecal samples were collected from each mouse in each group to obtain total genomic DNA, which was extracted using the QIAmp DNA Stool Mini Kit. The bacterial 16S rRNA gene (V3-V4 region) was amplified using the primer pair 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') in a polymerase chain reaction (PCR) thermocycler. The microbiota composition was determined according to the standard protocol, and the abundance of beneficial gut bacteria in the IMG group was increased by 2-fold ( Figure 19 ).

[0078] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A prebiotic hydrogel-coated nanomotor sustained-release preparation, characterized in that, Comprising: A nanomotor, which comprises mesoporous silica nanoparticles, amino groups modified on the surface of the mesoporous silica nanoparticles, and a manganese oxide layer asymmetrically coated on the surface of the mesoporous silica nanoparticles; A prebiotic hydrogel, which wraps the nanomotor; At least one active ingredient, which is loaded in the nanomotor and / or the prebiotic hydrogel and is used for treating inflammation or regulating the intestinal flora.

2. The sustained-release preparation of a prebiotic hydrogel-coated nanomotor according to claim 1, characterized in that: The prebiotic hydrogel is one or a combination of inulin, fructooligosaccharide, and galactooligosaccharide.

3. A prebiotic hydrogel-coated nanomotor sustained-release preparation according to claim 1 and a preparation method thereof, characterized in that: The active ingredient includes a traditional Chinese medicine monomer, an anti-inflammatory drug, an antioxidant, or a combination thereof.

4. A prebiotic hydrogel-encapsulated nanomotor sustained-release preparation according to claim 1 and a preparation method thereof, characterized in that: The traditional Chinese medicine monomer is glycyrrhizic acid or its derivative.

5. A prebiotic hydrogel-coated nano-motor sustained-release preparation according to claim 1 and a preparation method thereof, characterized in that: The manganese oxide layer is formed by an oxidative etching reaction and is used to generate gas in response to reactive oxygen species to drive the movement of the nanomotor.

6. A method for preparing a sustained-release preparation as described in any one of claims 1-5, characterized in that, Including the following steps: S1. Prepare amino-functionalized mesoporous silica nanoparticles; S2. Form a manganese oxide layer on the surface of the amino-functionalized mesoporous silica nanoparticles to obtain a nanomotor; S3. Mix the nanomotor with the active ingredient so that the active ingredient is loaded in the nanomotor; S4. Combine the nanomotor loaded with the active ingredient with the prebiotic hydrogel to form a sustained-release preparation that wraps the nanomotor.

7. A prebiotic hydrogel-encapsulated nanomotor sustained-release preparation and its preparation method according to claim 6, characterized in that: In the step S2, the manganese oxide layer is formed by one of the Pickering emulsion method and the oxidative etching method.

8. A prebiotic hydrogel-encapsulated nanomotor sustained-release preparation according to claim 6 and a preparation method thereof, characterized in that: In the step S4, the prebiotic hydrogel is dissolved by heating and then mixed with the nanomotor, and a gel structure is formed by cooling or standing.

9. Use of the sustained-release preparation according to any one of claims 1-5 in the preparation of a drug for treating inflammatory bowel disease, regulating the intestinal flora, or scavenging reactive oxygen species.

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