Sodium alginate hydrogel embedded bionic nanoparticles as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to sodium alginate hydrogel embedded bionic nanoparticles as well as a preparation method and application thereof. According to the nano-particle drug carrier and the preparation method thereof, the surfaces of nano-particles are modified with platelet membranes to form bionic nano-particles with inflammation targeting, and the bionic nano-particles are used for treating ulcerative colitis and remarkably improving the treatment effect; meanwhile, the bionic nano-particles are fused into the sodium alginate hydrogel, a layer of protective barrier is provided for the medicine, corrosion of gastric acid in the oral taking process is effectively resisted, stimulation of the medicine to gastrointestinal tracts is relieved, and the comfort level of a patient is improved.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a sodium alginate hydrogel-embedded bionic nanoparticle and a preparation method and application thereof. Background Art

[0002] Ulcerative colitis is a chronic nonspecific intestinal inflammatory disease. As one of the most common digestive diseases in clinical practice, ulcerative colitis greatly affects the quality of life of patients. Currently, there is no cure, and the main goal is to control symptoms, reduce recurrence, and prevent complications.

[0003] In recent years, the use of traditional Chinese medicine to harmonize qi and blood and nourish the five internal organs to achieve the purpose of whitening and removing freckles has become increasingly popular in the treatment of treatment. However, with the acceleration of the pace of modern life and the increasing requirements for the convenience, stability and bioavailability of drugs, the limitations of traditional Chinese medicine decoctions have become increasingly prominent. Due to the cumbersome decoction process, the difficulty in preservation and mildew, the inconvenience of carrying and the large dosage, it not only affects the patient's experience, but also limits the widespread application of Chinese medicine in the modern medical system. In addition, the chemical composition of Chinese medicine compound is complex, especially the active ingredient is the main material basis for the compound preparation to exert its efficacy, which is closely related to the prescription principle or compatibility theory of traditional Chinese medicine. It is this complexity that has brought challenges to the study of the compatibility of Chinese medicine compound in vivo.

[0004] Paeonia lactiflora total glucosides (Pa) is an effective ingredient extracted from the root of peony, mainly obtained by water extraction or ethanol extraction. Glycyrrhizic acid (GA) is the main active ingredient of licorice, with extensive and strong biological activities, such as anti-inflammatory, anti-allergic, anti-tumor, anti-viral, liver protection, and anti-ulcer. Due to its large relative molecular mass and high polarity, glycyrrhizic acid is difficult to be absorbed in its original form after oral administration. In order to be effectively utilized, glycyrrhizic acid needs to be hydrolyzed by bacteria in the gastrointestinal tract and converted into glycyrrhetinic acid before it can be absorbed into the blood. Therefore, whether glycyrrhizic acid is administered orally or by injection, it is mainly absorbed in the body in the form of its active metabolite, glycyrrhetinic acid, and distributed to various tissues and organs.

[0005] Therefore, there is an urgent need for a solution that can overcome the limitations of traditional Chinese medicine decoctions, improve drug bioavailability, and accurately deliver active ingredients to the lesions of ulcerative colitis. Summary of the invention

[0006] The purpose of the present invention is to provide a sodium alginate hydrogel-embedded bionic nanoparticle and a preparation method and application thereof. The method provided by the present invention not only overcomes the limitations of traditional Chinese medicine decoctions in the treatment of ulcerative colitis, but also improves the effective utilization rate of the drug.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] First, the present invention provides a bionic nanoparticle, wherein the bionic nanoparticle comprises paeoniflorin-glycyrrhizic acid.

[0009] Preferably, the paeoniflorin-glycyrrhizic acid is modified by platelets.

[0010] Second, the present invention also provides a method for preparing bionic nanoparticles, which comprises the following steps:

[0011] S1: Preparation of Pa-GA NPs

[0012] Paeoniflorin-glycyrrhizic acid Pa-GA and PLGA-PEG were weighed and dispersed in dichloromethane in a weight ratio of (0.1-1):1, and homogenized under high pressure in an ice bath for 3-5 minutes to obtain a primary nanocrystal suspension;

[0013] The primary nanocrystal suspension is added to an external aqueous phase 0.1-3% PVA aqueous solution, shaken, and homogenized under high pressure in an ice bath for 3-5 minutes to obtain a nanocrystal suspension;

[0014] Adding the nanocrystal suspension to a 0.1-3% PVA aqueous solution, stirring under ventilation, to obtain a nanoemulsion;

[0015] Centrifuging the nanoemulsion at high speed and washing to obtain Pa-GANPs;

[0016] S2: Preparation of platelet membrane-modified biomimetic Pa-GANPs

[0017] Platelet membrane (PM) was prepared by repeated freeze-thaw method;

[0018] The Pa-GANPs prepared in step S1 were dissolved in PBS buffer, a DMSO solution containing PM was added, and the mixture was ultrasonically treated in an ice bath, and freeze-dried to obtain PM@Pa-GANPs;

[0019] Preferably, in step S1, the mass ratio of PLGA-PEG to dichloromethane is 1:20-1:10; the volume ratio of the aqueous solution of PVA to dichloromethane is (1-4):1; the volume ratio of the nanosuspension to the aqueous solution of PVA is 1:(1-10); and the PVA is a PVA with a deacetylation degree of 85%-95%.

[0020] Preferably, in step S1, high-speed centrifugation is performed at 0°C and 12000 g for 20 min.

[0021] Preferably, in step S2, the step of extracting PM is as follows: blood containing heparin or EDTA anticoagulant is centrifuged at 1500-2500 r / min for 10 min to obtain platelet-rich plasma, and then centrifuged at 12000-18000 r / min for 8-12 min to separate platelets. The obtained platelet sample is repeatedly frozen and thawed three times, and then homogenized on ice for 1 min to obtain vesicles containing platelet membranes.

[0022] Preferably, in step S2, the weight-to-volume ratio of Pa-GA NPs to PBS buffer is (0.01-1):1; the volume ratio of the PM-containing DMSO solution to PBS buffer is (0.1-1):1.

[0023] Preferably, in step S3, the volume ratio of the sodium alginate solution to the calcium chloride solution is (1-10):1.

[0024] Third, the present invention provides a hydrogel-embedded bionic nanoparticle, including the platelet-modified bionic nanoparticle according to claim 2.

[0025] The invention prepares the bionic nanoparticles embedded in sodium alginate hydrogel in order to resist the erosion of gastric acid when taken orally.

[0026] Fourthly, the present invention also provides a method for preparing hydrogel-embedded bionic nanoparticles, comprising the method for preparing bionic nanoparticles according to claim 3, and further comprising the following preparation method:

[0027] 0.5-2% CaCl2 solution is added dropwise to 0.1-3% sodium alginate solution, and the volume ratio of sodium alginate solution to calcium chloride solution is (1-10):1, and stirred. After stirring for 10 minutes, a hydrogel is formed. The PM@Pa-GANPs prepared in step S2 are mixed with the sodium alginate hydrogel to obtain bionic nanoparticles with a sodium alginate hydrogel (sam) loaded with a platelet membrane coating.

[0028] Fifth, the present invention also provides an application of bionic nanoparticles in oral drugs related to the treatment of ulcerative colitis.

[0029] Beneficial effects:

[0030] (1) The present invention is designed to use the effective ingredients of Chinese medicine, total glycosides of paeonia lactiflora and glycyrrhizic acid, which have significant anti-inflammatory and repair effects in the treatment of ulcerative colitis. They can not only effectively reduce the inflammatory response of the intestinal mucosa, relieve symptoms such as abdominal pain and diarrhea, but also promote the healing and regeneration of the intestinal mucosa and restore the normal physiological function of the intestine.

[0031] (2) The present invention achieves inflammation targeting of nanoparticles by modifying the platelet membrane on the surface of nanoparticles, enabling them to actively and accurately identify and bind to the site of inflammation. The drug can act directly and efficiently on the lesion area, thereby significantly reducing systemic drug side effects. This targeted delivery method greatly improves the treatment effect.

[0032] (3) The present invention uses sodium alginate hydrogel as a drug carrier, which effectively resists the corrosion of gastric acid during oral administration and ensures that the drug maintains its integrity and activity before reaching the absorption site such as the small intestine. The sustained release properties of sodium alginate hydrogel help to prolong the release time of the drug, so that the drug maintains an effective concentration in the body for a longer period of time, thereby providing a more lasting therapeutic effect.

[0033] (4) The present invention integrates nanotechnology and hydrogel technology, so that the nanoparticles not only have high targeting and adhesion, and can accurately deliver drug components to the lesions of ulcerative colitis, but also achieve continuous and stable release of drug components through the sustained release mechanism of hydrogel, thereby greatly enhancing the therapeutic effect.

[0034] (5) The preparation method of the present invention is simple, practical and easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0036] Figure 1 The schematic diagram of the preparation method of sodium alginate hydrogel loaded bionic nanoparticle drug carrier; wherein A is the preparation process of bionic nano drug carrier, and B is the process of sodium alginate hydrogel loaded bionic nano drug carrier.

[0037] Figure 2 Schematic diagram of the inflammatory cell targeting experiment of platelet membrane-modified bionic nanoparticles; A is the fluorescence intensity, and B is the cell imaging using an inverted fluorescence microscope.

[0038] Figure 3 Schematic diagram of whole body and colon tissue imaging of mice after oral administration of hydrogels at different times.

[0039] From left to right are blank control group, control group, and experimental group.

[0040] Figure 4Schematic diagram of the therapeutic effect on colitis mice 4 hours after oral administration. The upper figure is a picture of the colon of each treatment group; the lower figure is a comparison of the colon length of each treatment group.

[0041] Figure 5 Schematic diagram of the therapeutic effect on colitis mice 8 hours after oral administration. The upper figure is a picture of the colon of each treatment group; the lower figure is a comparison of the colon length of each treatment group.

[0042] Figure 6 Schematic diagram of the therapeutic effect on colitis mice 24 hours after oral administration. The upper figure is a picture of the colon of each treatment group; the lower figure is a comparison of the colon length of each treatment group.

[0043] Figure 7 A is a schematic diagram of the changes and processes of inflammatory factors in colon tissue of each treatment group. A is a schematic diagram of the induction of colitis model and the timeline of drug intervention treatment in each group; B is a schematic diagram of the changes of inflammatory factors in colon tissue of each group.

[0044] Figure 8 Schematic diagram of pathological sections of mice in the hydrogel group 7 days after oral administration. DETAILED DESCRIPTION

[0045] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] In one aspect, the present invention provides a biomimetic nanoparticle.

[0047] In one aspect, the present invention also provides a method for preparing bionic nanoparticles, comprising the following steps:

[0048] S1: Preparation of Pa-GA NPs;

[0049] S2: Preparation of platelet membrane-modified biomimetic Pa-GANPs;

[0050] Further, in step S1, paeoniflorin-glycyrrhizic acid Pa-GA and PLGA-PEG are weighed in a weight ratio of (0.1-1):1, dispersed in dichloromethane, and homogenized under high pressure in an ice bath for 3-5 minutes to obtain a primary nanocrystal suspension;

[0051] The primary nanocrystal suspension is added to an aqueous solution of 0.1-3% PVA in the external aqueous phase, shaken, and homogenized under high pressure in an ice bath for 3-5 minutes to obtain a nanocrystal suspension; preferably, Pa-GA and PLGA-PEG are weighed in a weight ratio of 0.5:1, dispersed in dichloromethane, and homogenized under high pressure for 4 minutes. The primary nanocrystal suspension is added to an aqueous solution of 2% PVA in the external aqueous phase, shaken, and homogenized under high pressure in an ice bath for 4 minutes.

[0052] The volume ratio of the PVA aqueous solution to dichloromethane is (1-4):1, preferably the volume ratio of the PVA aqueous solution to dichloromethane is 2:1;

[0053] Further, in step S2, the step of extracting PM is: centrifuging the blood containing heparin or EDTA anticoagulant at 1500-2500r / min for 10 minutes to obtain platelet-rich plasma, and then centrifuging at 12000-18000r / min for 8-12 minutes to separate platelets. After the obtained platelet sample is repeatedly frozen and thawed three times, homogenized on ice for 1 minute to obtain vesicles containing platelet membranes. Preferably, the centrifugal speed is 2000r / min, centrifuged for 10 minutes to obtain platelet-rich plasma, and then centrifuged at 16000r / min for 10 minutes to separate platelets.

[0054] On the other hand, the present invention provides a hydrogel-embedded biomimetic nanoparticle, comprising the platelet-modified biomimetic nanoparticle according to claim 2.

[0055] The present invention also provides a method for preparing hydrogel-embedded bionic nanoparticles, comprising the method for preparing bionic nanoparticles according to claim 3, and further comprising the following preparation method:

[0056] 0.5-2% CaCl2 solution is added dropwise to 0.1-3% sodium alginate solution, and the volume ratio of sodium alginate solution to calcium chloride solution is (1-10):1, and stirred. After stirring for 10 minutes, a hydrogel is formed. The PM@Pa-GANPs prepared in step S2 are mixed with the sodium alginate hydrogel to obtain bionic nanoparticles with a sodium alginate hydrogel (sam) loaded with a platelet membrane coating.

[0057] Further, 0.5-2% CaCl2 solution is added dropwise to 0.1-3% sodium alginate solution, stirred for 10 minutes, to form a hydrogel. Preferably, 1% CaCl2 solution is added dropwise to 2% sodium alginate solution.

[0058] On the other hand, the present invention provides the use of bionic nanoparticles in oral drugs related to the treatment of ulcerative colitis.

[0059] Example 1

[0060] Preparation of Pa-GA NPs

[0061] 1) Pa-GA and PLGA-PEG were accurately weighed in a weight ratio of 0.5:1 and dispersed in 5 ml of dichloromethane solvent. The dispersion was homogenized for 4 min using a high-pressure homogenizer under ice bath conditions to obtain a uniform primary nanocrystal suspension;

[0062] 2) taking a 2% PVA aqueous solution as the external aqueous phase, adding the primary nanocrystal suspension prepared in step 1) to the external aqueous phase, and shaking the mixture in an ice bath, and then again using a high-pressure homogenizer to perform high-pressure homogenization on the mixture for 4 minutes to form a nanocrystal suspension.

[0063] 3) adding an aqueous solution of PVA to the nanocrystal suspension obtained in step 2), placing the mixture in a fume hood, and stirring the mixture with a magnetic stirrer for 5 hours to obtain a nanoemulsion;

[0064] 4) The nanoemulsion obtained in step 3) is transferred to a centrifuge tube and centrifuged at high speed using a centrifuge. After centrifugation, the supernatant is carefully discarded and the precipitate is retained. The precipitate is washed with ultrapure water and washed more than three times to remove residual impurities such as PVA and dichloromethane. After washing, the precipitate is dried, and freeze drying or vacuum drying can be used. After drying, the solid obtained is collected, which is the Pa-GA-PLGA-PEG nanoparticles (Pa-GA NPs) with integrated paeoniflorin-glycyrrhizic acid.

[0065] Example 2

[0066] Preparation of platelet membrane-modified biomimetic Pa-GANPs

[0067] Platelet membrane (PM) was prepared by repeated freezing and thawing: blood containing heparin or EDTA anticoagulant was centrifuged at 2000r / min for 10min to obtain platelet-rich plasma, and then centrifuged at 15000r / min for 10min to separate platelets. The obtained platelet sample was repeatedly frozen and thawed three times, and homogenized on ice for 1min to obtain vesicles containing platelet membrane;

[0068] The Pa-GANPs prepared in step S1 were dissolved in PBS buffer, and a DMSO solution containing PM was added, and the mixture was ultrasonically treated in an ice bath, and then freeze-dried to obtain PM@Pa-GANPs. Figure 1 -A.

[0069] Example 3

[0070] Preparation of Sodium Alginate Hydrogel Loaded with Biomimetic Nano-drug

[0071] 1% CaCl2 solution was added dropwise to 2% sodium alginate solution and stirred for 10 min to form a hydrogel. PM@Pa-GANPs prepared in step S2 were mixed with sodium alginate hydrogel to obtain a hydrogel loaded with biomimetic nano drug carriers. Figure 1 -B.

[0072] Example 4

[0073] Experimental verification of the inflammatory cell targeting of platelet membrane-modified biomimetic nanoparticles

[0074] Under appropriate culture conditions, normal cells and inflammatory cells induced by LPS (lipopolysaccharide) were cultured separately. The bionic PM@Pa-GANPs hydrogel prepared in Example 3 (loaded with coumarin 6 as a fluorescent marker) was co-incubated with the above two cells for two hours. After the incubation, the cells were washed with PBS buffer to remove unbound nanoparticles. The washed cells were resuspended in PBS buffer, and then the resuspended cell sample was injected into a flow cytometer to detect the fluorescence intensity in 10,000 cells. At the same time, as a control, the fluorescence intensity of ordinary PLGA nanoparticles loaded with the same drug (i.e., nanoparticles without platelet modification and without hydrogel) in the cells was also detected. The test results are as follows. Figure 2 shown.

[0075] The experimental results showed that: through flow cytometry detection, Figure 2 -A, the biomimetic nanoparticles modified with platelet membranes have significantly increased cell targeting in both normal cells and LPS-induced inflammatory cells compared to ordinary nanoparticles. Figure 2 -B, it can be seen that the distribution of biomimetic nanoparticles modified with platelet membranes in cells is significantly increased.

[0076] Example 5

[0077] Validation of the effectiveness and bioavailability of hydrogels as drug carriers

[0078] Eight-week-old male C57 / 6J mice were purchased from Guangdong Animal Experiment Center and randomly divided into three groups: blank control group, experimental group and control group. The blank control group did not receive any treatment; the experimental group received oral administration of a biomimetic nano-drug carrier solution containing sodium alginate hydrogel (hydrogel group); and the control group received oral administration of a solution containing only biomimetic nano-drug carriers (original drug group). Figure 3-5 The drug solution was administered to mice by gavage. At different time points (4h, 8h, 24h) after administration, the whole body of the mice was imaged using a fluorescence microscope, and the distribution of the fluorescent dye in the mice was observed and recorded, as shown in FIG. Figure 3-5As shown in the figure, since the fluorescence of the drug is very weak, the fluorescent dye indocyanine green is co-loaded into the nano drug carrier as its fluorescent marker.

[0079] Depend on Figure 3-5 The experimental results show that hydrogel can significantly prolong the enrichment and retention time of bionic nano-drug carriers in the mouse colon, thereby verifying the effectiveness and bioavailability of hydrogel as a drug carrier.

[0080] After the mice were adapted for one week, the drinking water was replaced with 2.5% DSS (dextran sulfate sodium salt) to induce the ulcerative colitis model. After the 7th day, the drinking water was replaced with normal drinking water. Figure 7 As shown in A. Starting from the first day of membrane formation, mice in the experimental group and the control group were gavaged with drugs at a dose of 10 mg / kg every day. At 4 hours, 8 hours, and 24 hours after administration, the whole body of the mice was imaged using a fluorescence microscope to observe and record the distribution of the fluorescent dye in the mice. Figure 6 , Figure 7 B. After 48 hours, i.e., on the 10th day, the mice were euthanized and colon tissue samples were collected. Pathological sections of the collected colon tissue were prepared and observed under a microscope, as shown in FIG. Figure 8 shown.

[0081] Depend on Figure 6 , Figure 7 The experimental results show that after 7 days of oral administration, the shortening of the colon in mice with colitis was significantly inhibited, and the hydrogel nanomedicine significantly inhibited the secretion of colitis factors.

[0082] Depend on Figure 8 The experimental results show that after 7 days of oral administration, the colon tissue pathological sections of mice in the hydrogel group showed that compared with mice that did not receive hydrogel treatment, the degree of colon inflammation was significantly reduced and the tissue structure was more intact.

[0083] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A biomimetic nanoparticle, characterized in that: The bionic nanoparticles include paeoniflorin-glycyrrhetinic acid.

2. The biomimetic nanoparticle according to claim 1, characterized in that: The paeoniflorin-glycyrrhizic acid is modified by platelets.

3. A method for preparing biomimetic nanoparticles, characterized in that: The preparation method comprises the following steps: S1: Preparation of Pa-GA NPs Paeoniflorin-glycyrrhizic acid Pa-GA and PLGA-PEG were weighed and dispersed in dichloromethane in a weight ratio of (0.1-1):1, and homogenized under high pressure in an ice bath for 3-5 minutes to obtain a primary nanocrystal suspension; The primary nanocrystal suspension is added to a 0.1-3% PVA aqueous solution, shaken, and homogenized under high pressure in an ice bath for 3-5 minutes to obtain a nanocrystal suspension; Adding the nanocrystal suspension to a 0.1-3% PVA aqueous solution, stirring under ventilation, to obtain a nanoemulsion; The nanoemulsion is centrifuged at high speed and washed to obtain Pa-GANPs; S2: Preparation of platelet membrane-modified biomimetic Pa-GANPs Platelet membrane (PM) was prepared by repeated freeze-thaw method; The Pa-GANPs prepared in step S1 were dissolved in PBS buffer, and a DMSO solution containing PM was added, and the mixture was ultrasonically treated under ice bath conditions, and then freeze-dried to obtain PM@Pa-GANPs.

4. The preparation method according to claim 3, characterized in that: In the step S1, the mass ratio of PLGA-PEG to dichloromethane is 1:20-1:10; the volume ratio of the aqueous solution of PVA to dichloromethane is (1-4):1; the volume ratio of the nanosuspension to the aqueous solution of PVA is 1:(1-10); and the PVA has a deacetylation degree of 85%-95%.

5. The preparation method according to claim 3, characterized in that: In step S1, high-speed centrifugation is performed at 0°C, 12000 g, and centrifugation for 20 min.

6. The preparation method according to claim 3, characterized in that: In step S2, the step of extracting PM is as follows: blood containing heparin or EDTA anticoagulant is centrifuged at 1500-2500 r / min for 10 min to obtain platelet-rich plasma, and then centrifuged at 12000-18000 r / min for 8-12 min to separate platelets. The obtained platelet sample is repeatedly frozen and thawed three times, and then homogenized on ice for 1 min to obtain vesicles containing platelet membranes.

7. The preparation method according to claim 3, characterized in that: In the step S2, the weight-to-volume ratio of Pa-GA NPs to PBS buffer is (0.01-1):1; the volume ratio of the PM-containing DMSO solution to PBS buffer is (0.1-1):

1.

8. A biomimetic nanoparticle embedded in a hydrogel, characterized in that: Including the platelet-modified bionic nanoparticles as described in claim 2.

9. A method for preparing hydrogel-embedded biomimetic nanoparticles, characterized in that: The preparation method includes the biomimetic nanoparticle preparation method according to claim 3, and also includes the following preparation method: 0.5-2% CaCl2 solution is added dropwise to 0.1-3% sodium alginate solution, and the volume ratio of sodium alginate solution to calcium chloride solution is (1-10):1, and stirred. After stirring for 10 minutes, a hydrogel is formed. The PM@Pa-GA NPs prepared in step S2 are mixed with the sodium alginate hydrogel to obtain bionic nanoparticles with a sodium alginate hydrogel (sam) loaded with a platelet membrane coating.

10. Use of the biomimetic nanoparticles according to any one of claims 1 to 2 or claim 8 in oral medications for treating ulcerative colitis.

Images