PDRN-loaded tea vesicle-like nanoparticles as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nanoparticles, and particularly discloses PDRN-loaded tea vesicle-like nanoparticles as well as a preparation method and application thereof, and the preparation method comprises the following steps: extracting the tea vesicle-like nanoparticles; preparing tea leaf vesicle-like nanoparticles loaded with polydeoxyribonucleotide; and characterizing the polydeoxyribonucleotide-loaded tea vesicle-like nanoparticles. According to the PDRN-loaded tea vesicle-like nano-particles as well as the preparation method and the application thereof, the PDRN-loaded tea vesicle-like nano-particles have a stable lipid bilayer structure in gastrointestinal simulation fluid, can be orally taken for treating diseases, provides a new strategy for oral application of PDRN, and has a relatively good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of nanoparticles, and in particular to tea vesicle-like nanoparticles loaded with PDRN, and a preparation method and application thereof. Background Art

[0002] Polydeoxyribonucleotide (PDRN) is a bioactive natural compound used in regenerative medicine. This compound contains a mixture of deoxyribonucleotides with molecular weights ranging from 50 to 1,500 KDa, derived from a controlled purification and sterilization process of sperm DNA from salmon trout (Oncorhynchus mykiss) or salmon (Oncorhynchus keta). This procedure ensures the absence of active proteins and peptides that may cause immune responses. PDRN can exert multiple effects such as tissue repair, anti-ischemia and anti-inflammatory effects by activating A2 receptors and DNA salvage pathways, and is therefore widely used in skin repair, ulcer healing and other fields.

[0003] In existing studies, the application of PDRN is concentrated on injection and transdermal administration. Injection includes intradermal injection, subcutaneous tissue injection and intraperitoneal injection. These methods can ensure that PDRN directly reaches deep into the body and plays the role of promoting cell regeneration, anti-inflammatory and antioxidant effects. However, the injection method has low product popularity and adverse reactions such as pain. The efficacy of transdermal administration is proportional to the surface area covered by the drug and its fat solubility. PDRN is a hydrophilic macromolecule and it is difficult to pass through the skin to exert its efficacy. Oral administration has the advantages of convenience, non-sterile production requirements and few side effects, but oral PDRN still faces challenges. PDRN has unique physicochemical properties such as high molecular weight, hydrophilicity and negative charge, and encounters problems such as instability and low cellular uptake during oral delivery. In addition, the gastrointestinal tract (GIT) is naturally designed for compound digestion and processing, with harsh pH conditions, enzymes, thick mucus layers and various intestinal cells, which provide physical and chemical barriers, mucus barriers and epithelial barriers for the absorption of PDRN.

[0004] The main components of tea are tea polyphenols, amino acids, alkaloids, sugars, proteins, pectin, aromatic substances, enzymes and organic acids, which have multiple functions such as antioxidant, anti-inflammatory, antibacterial, cardiovascular protection, anti-sugar and anti-obesity properties. Tea vesicle-like nanoparticles (EVs) have excellent physical and chemical properties such as inherent stability, safety and low immunogenicity, as well as the ability to cross biological barriers, and can be used as oral therapeutic agents. In addition, tea vesicle-like nanoparticles have a spherical structure composed of phospholipid bilayers, which can embed or adsorb various drugs between the bilayers, or encapsulate different types of drugs in the inner cavity, thus becoming an excellent oral drug carrier.

[0005] The application of PDRN in existing literature is limited to injection and transdermal administration. Patient compliance of injection is poor and the aseptic requirements are high. The drug absorption capacity of transdermal administration is poor, which greatly limits the application of PDRN. In existing literature, drug carriers loaded with PDRN use emulsifiers or low-toxic reagents, which poses a safety risk.

[0006] To this end, the present invention proposes a method for preparing tea vesicle-like nanoparticles loaded with PDRN to solve the problems raised by the background technology. Summary of the invention

[0007] The purpose of the present invention is to provide tea vesicle-like nanoparticles loaded with PDRN, a preparation method and an application thereof. The tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides have a stable lipid bilayer structure in a gastrointestinal simulated fluid, can be orally used to treat diseases, provide a new strategy for the oral application of PDRN, and have good application prospects.

[0008] To achieve the above object, the present invention provides a method for preparing tea vesicle-like nanoparticles loaded with PDRN, comprising the following steps:

[0009] Step 1, extracting tea vesicle-like nanoparticles, washing and chopping tea leaves to squeeze juice, collecting the initial product in a centrifuge bottle, performing differential centrifugation, discarding the precipitate after centrifugation, taking the supernatant, adding the supernatant to an ultracentrifuge and centrifuging to obtain tea vesicle-like nanoparticles;

[0010] Step 2, preparing tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides, mixing polydeoxyribonucleotides with tea vesicle-like nanoparticles, incubating with low-power ultrasound at 37° C., 0-100 rpm, for 1-3 hours to promote membrane fusion; adding the mixed solution to an ultracentrifuge for centrifugation to remove free polydeoxyribonucleotides;

[0011] Step three, characterize the tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides, determine the encapsulation efficiency and drug loading of polydeoxyribonucleotides in the tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides by an indirect method; use a dynamic light scattering particle size analyzer to determine the hydrodynamic particle size and polydispersity index of the tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides; use a nanoparticle tracking analyzer to detect the particle size of the tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides; and observe the structure of the tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides by transmission electron microscopy.

[0012] Preferably, in step 1, the centrifugation parameters of the differential centrifugation are 1000×g for 10 min, 3000×g for 20 min, and 10000×g for 40 min.

[0013] Preferably, the centrifugation parameters in the ultracentrifuge are 130,000×g to 170,000×g for 60 to 90 min.

[0014] Preferably, in step 1, the specific operation of extracting tea vesicle-like nanoparticles is as follows: take 100g to 200g of tea, remove thick stems and yellow leaves and insect leaves, wash them with ultrapure water for 1 to 3 times, chop them and pour them into a juicer, add 200mL to 400mL of ultrapure water, and squeeze the juice for 60 to 120S; squeeze the squeezed mixture with an extruder, and collect the primary product in a centrifuge bottle; the primary product is balanced and placed in a high-speed centrifuge for differential centrifugation to remove large particles, dead cells and cell fragments, and the centrifugation parameters are 1000×g centrifugation for 10min, 3000×g centrifugation for 20min, and 10000×g centrifugation for 40min; after high-speed centrifugation, discard the precipitate, take the supernatant, add the supernatant to an ultracentrifuge, centrifuge at 130000×g to 170000×g for 60 to 90min to obtain tea extracellular vesicles, resuspend and collect them with a sodium chloride solution with a concentration of 0.9%, and store them at -80°C to avoid repeated freezing and thawing when taking them.

[0015] Preferably, in step 2, the specific operation of preparing tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides is as follows: polydeoxyribonucleotides and tea vesicle-like nanoparticles are mixed in a ratio of 1 to 8:1, low-power ultrasound, 10 to 30 seconds on, 30 to 50 seconds off, 4 to 8 cycles of drug loading; 37°C, 0 to 100 rpm, 1 to 3 hours of incubation to promote membrane fusion; the solution is added to an ultracentrifuge, and centrifuged at 130,000 × g to 170,000 × g for 60 to 90 minutes to remove free polydeoxyribonucleotides.

[0016] Preferably, in step three, the indirect method for determining the encapsulation efficiency and drug loading of polydeoxyribonucleotides in tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides is as follows: 200 μL to 500 μL of drug-loaded tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides are added to a 100 kD to 300 kD ultrafiltration centrifuge tube, centrifuged at 10000 rpm to 15000 rpm for 10 min to 30 min, and the absorbance of the filtrate at 260 nm is detected.

[0017] Preferably, in step three, the operation of using a dynamic light scattering particle size analyzer to determine the flow dynamics particle size and polydispersity index of tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides is as follows: take an appropriate amount of tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides and dissolve them in 1.5-2 mL of deionized water, the final concentration of the tea vesicle-like nanoparticles is 0.5-2 mg / mL, and use a dynamic light scattering particle size analyzer to determine the flow dynamics particle size and polydispersity index of the tea vesicle-like nanoparticles, and measure three times to obtain the average value.

[0018] Preferably, in step three, the operation of observing the structure of tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides by transmission electron microscopy is as follows: taking the mixed sample and dropping it on the carbon support film copper mesh, and letting it stand for 5 minutes; using filter paper to absorb the residual liquid from the edge of the copper mesh, adding 2% phosphotungstic acid to the copper mesh, dyeing at room temperature for 30 seconds, absorbing excess dye with filter paper, and placing it under a transmission electron microscope for observation after natural drying.

[0019] The tea vesicle-like nanoparticles loaded with PDRN are prepared by the above-mentioned preparation method.

[0020] The present invention also provides the application of tea vesicle-like nanoparticles loaded with PDRN, which is applied to the preparation of oral medicines for treating inflammatory bowel disease.

[0021] The advantages and beneficial effects of the tea vesicle-like nanoparticles loaded with PDRN and the preparation method and application of the present invention are:

[0022] The tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides of the present invention have a stable lipid bilayer structure in a gastrointestinal simulated fluid, can be taken orally for treating diseases, provide a new strategy for the oral application of PDRN, and have good application prospects.

[0023] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of extracting tea vesicle-like nanoparticles of the present invention;

[0025] Figure 2 TEM image of tea vesicle-like nanoparticles of the present invention;

[0026] Figure 3 is the particle size of the tea vesicle-like nanoparticles of the present invention after being incubated in gastrointestinal fluid for 2 hours;

[0027] Figure 4 is the PDI of the tea vesicle-like nanoparticles of the present invention after being incubated in gastrointestinal fluid for 2 hours;

[0028] Figure 5 Schematic diagram of the preparation of tea vesicle-like nanoparticles loaded with PDRN according to the present invention;

[0029] Figure 6 TEM image of tea vesicle-like nanoparticles loaded with PDRN according to the present invention;

[0030] Figure 7 The particle size of the tea vesicle-like nanoparticles loaded with PDRN in the present invention after incubation in gastrointestinal fluid for 2 hours;

[0031] Figure 8 It is the PDI of the tea vesicle-like nanoparticles loaded with PDRN of the present invention after being incubated in gastrointestinal fluid for 2 hours;

[0032] Fig. 9 TEM images of the tea vesicle-like nanoparticles loaded with PDRN according to the present invention after being incubated in different gastrointestinal fluids for 2 hours, wherein A is phosphate buffer, B is simulated gastric fluid, C is simulated small intestinal fluid, and D is simulated colon fluid;

[0033] Fig.10 This is a schematic diagram of the animal experiment of the present invention;

[0034] Fig.11 This is the weight change curve of mice of the present invention;

[0035] Fig.12 This is a disease activity index scoring diagram of the present invention;

[0036] Fig.13 This is a bar graph of the colon length of each group after the mice were dissected on the 8th day of the present invention;

[0037] Fig.14 These are H&E staining images of colon tissue sections of the present invention, wherein A is a blank group, B is a DSS group, C is an EV group, D is a PDRN group, and E is a PDRN-EV group. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0039] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0040] Unless otherwise defined, the reagents and equipment used in the present invention are commonly available from the market.

[0041] Example 1

[0042] The preparation method of PDRN-loaded tea vesicle-like nanoparticles (PDRN-EV) comprises the following steps:

[0043] Step 1: extract tea vesicle nanoparticles (EV), take 100g-200g tea (remove thick stems and yellow leaves and insect leaves), wash with ultrapure water 1-3 times; put it on a chopping board and chop it into pieces, pour it into a juicer, add 200mL-400mL ultrapure water, and squeeze the juice for 60-120S; squeeze the squeezed mixture with an extruder, and collect the primary product in a centrifuge bottle; the primary product is balanced and placed in a high-speed centrifuge for differential centrifugation to remove large particles, dead cells and cell debris. The centrifugation parameters are 1000×g for 10 min, 3000×g for 20 min, and 10000×g for 40 min. After high-speed centrifugation, discard the precipitate, take the supernatant, add the supernatant to an ultracentrifuge, and centrifuge at 130000×g to 170000×g for 60 to 90 min to obtain tea vesicle-like nanoparticles. Use an appropriate amount of 0.9% sodium chloride (NaCl) solution to resuspend and collect, store at -80°C, and avoid repeated freezing and thawing when taking out. Figure 1 shown.

[0044] Step 2: prepare tea vesicle nanoparticles (PDRN-EV) loaded with polydeoxyribonucleotide (PDRN), mix polydeoxyribonucleotide (PDRN) and tea vesicle nanoparticles (EV) in a ratio of 1 to 8:1, perform low-power ultrasound, on for 10 to 30 seconds, off for 30 to 50 seconds, and perform 4 to 8 cycles of drug loading; incubate at 37°C, 0 to 100 rpm, for 1 to 3 hours to promote membrane fusion; add the solution to an ultracentrifuge, centrifuge at 130,000 × g to 170,000 × g for 60 to 90 minutes to remove free PDRN, such as Figure 5 shown.

[0045] Step three, characterize the tea vesicle-like nanoparticles (PDRN-EV) loaded with polydeoxyribonucleotides, and indirectly determine the encapsulation efficiency (EE%) and drug loading (LC%) of PDRN in PDRN-EV. Take 200μL~500μL of drug-loaded PDRN-EV and add it to a 100kD~300kD ultrafiltration centrifuge tube, centrifuge at 10000rpm~15000rpm for 10min~30min, and use Nanodrop to detect the absorbance of the filtrate at 260nm. For the determination of size and polydispersity index, take an appropriate amount of tea vesicle-like nanoparticles loaded with PDRN and dissolve them in 1.5~2mL deionized water (the final concentration of tea vesicle-like nanoparticles is 0.5~2mg / mL), and use a dynamic light scattering particle size analyzer to determine the hydrodynamic particle size and polydispersity index of tea vesicle-like nanoparticles. Determine three times and calculate the average value.

[0046] For NTA particle size analysis of PDRN-EVs, the prepared PDRN-EVs were injected into the Nanosight nanoparticle tracking analyzer for detection.

[0047] The structure was observed by transmission electron microscopy (TEM). The mixed sample was dripped onto the carbon support film copper mesh and allowed to stand for 5 minutes. The residual liquid was removed from the edge of the copper mesh with filter paper. 2% phosphotungstic acid was dripped onto the copper mesh and dyed at room temperature for 30 seconds. The excess dye was removed with filter paper. After drying naturally, the sample was placed under a transmission electron microscope for observation. Figure 6 As shown, the tea vesicle-like nanoparticles loaded with PDRN have a complete nanostructure, indicating that the ultrasonic drug loading process did not destroy the structure of the tea vesicle-like nanoparticles.

[0048] The tea vesicle-like nanoparticles loaded with polydeoxyribonucleotide prepared by the above-mentioned preparation method are used for preparing oral drugs for treating inflammatory bowel disease.

[0049] Table 1 shows the drug loading and encapsulation efficiency results of tea vesicle-like nanoparticles loaded with PDRN.

[0050] Table 1 Drug loading and encapsulation efficiency results

[0051]

[0052] It can be seen from Table 1 that when the feed ratio of tea vesicle-like nanoparticles and PDRN is 1:4.48, the encapsulation efficiency is 16% and the drug loading is 41.5%, achieving effective encapsulation of PDRN in tea vesicle-like nanoparticles.

[0053] Example 2

[0054] (1) Detect the protein concentration in tea vesicle-like nanoparticles.

[0055] The sample concentration was determined by the BCA kit. First, the protein standard and BCA working solution were prepared. Then, the protein concentration standard and tea vesicle-like nanoparticle stock solution were added to the 96-well ELISA plate at different concentrations. The BCA working solution was added in proportion. Then, the plate was incubated at 37°C for 30 minutes. The optical density (OD) value was measured at 562nm using an ELISA reader. Based on the measured OD value, combined with the standard curve, the dilution multiple of each experimental group was used to obtain the corresponding protein concentration of each group. Three parallel runs were set for each group to calculate the average value to reduce experimental errors.

[0056] (2) Characterization of tea vesicle-like nanoparticles.

[0057] For the determination of size and polydispersity index, an appropriate amount of tea vesicle-like nanoparticles was dissolved in 1.5-2 mL of deionized water (the final concentration of tea vesicle-like nanoparticles was 0.5-2 mg / mL). A dynamic light scattering particle size analyzer was used to determine the hydrodynamic particle size and polydispersity index of the tea vesicle-like nanoparticles. The average value was calculated by three measurements.

[0058] The structure was observed by transmission electron microscopy (TEM). The mixed sample was dripped onto the carbon support film copper mesh and allowed to stand for 5 minutes. The residual liquid was removed from the edge of the copper mesh with filter paper. 2% phosphotungstic acid was dripped onto the copper mesh and dyed at room temperature for 30 seconds. The excess dye was removed with filter paper. After drying naturally, the sample was placed under a transmission electron microscope for observation. Figure 2 As shown, it shows that tea vesicle-like nanoparticles have a cavity structure and a lipid bilayer structure, and are suitable as drug delivery carriers.

[0059] (3) The stability of tea vesicle-like nanoparticles in simulated gastrointestinal fluid.

[0060] Preparation of simulated gastrointestinal fluid: 80mM hydrochloric acid, 35mM sodium chloride, 0.3% pepsin, add deionized water to make up to 100mL, pH 1.2; Preparation of simulated small intestinal fluid: 15mM sodium hydroxide, 50mM potassium dihydrogen phosphate, 1% pancreatic enzyme, add deionized water to make up to 100mL, pH 6.8; Preparation of simulated colonic fluid: 559mg dipotassium hydrogen phosphate and 41mg potassium dihydrogen phosphate, add deionized water to make up to 100mL, pH 7.8.

[0061] The tea vesicle-like nanoparticles were diluted with phosphate buffer (PBS), simulated gastrointestinal fluid, simulated small intestinal fluid, and simulated colonic fluid to a concentration of 0.5-2 mg / mL, incubated at 37°C and 200 rpm for 1-4 hours, and the flow dynamics particle size and polydispersity index of the tea vesicle-like nanoparticles were measured using a dynamic light scattering particle size analyzer. The average value was calculated by three measurements; the structure was observed by transmission electron microscopy. Figure 3 As shown, it shows that there is no significant difference in the particle size of tea vesicle-like nanoparticles in different solutions, indicating that the structure of tea vesicle-like nanoparticles in different solutions remains stable and has the ability to resist digestion by gastrointestinal fluid. Figure 4 As shown, the polydispersity index of tea vesicle-like nanoparticles in different solutions is less than 0.3, indicating that tea vesicle-like nanoparticles have colloidal stability in different solutions.

[0062] Example 3

[0063] Stability of tea vesicle-like nanoparticles loaded with PDRN in simulated gastrointestinal fluid.

[0064] ① Preparation of simulated gastrointestinal fluid: 80mM hydrochloric acid, 35mM sodium chloride, 0.3% pepsin, add deionized water to make up to 100mL, pH 1.2; Preparation of simulated small intestinal fluid: 15mM sodium hydroxide, 50mM potassium dihydrogen phosphate, 1% pancreatic enzyme, add deionized water to make up to 100mL, pH 6.8; Preparation of simulated colonic fluid: 559mg dipotassium hydrogen phosphate and 41mg potassium dihydrogen phosphate, add deionized water to make up to 100mL, pH 7.8.

[0065] ② The tea vesicle nanoparticles loaded with PDRN were diluted with phosphate buffer (PBS), simulated gastrointestinal fluid, simulated small intestinal fluid, and simulated colonic fluid to a concentration of 0.5-2 mg / mL, incubated at 37°C and 200 rpm for 1-4 hours, and the hydrodynamic particle size and polydispersity index of the tea vesicle nanoparticles were measured using a dynamic light scattering particle size analyzer. The average value was calculated by three measurements; the structure was observed by transmission electron microscopy. Figure 7 As shown in the figure, there is no significant difference in the particle size of tea vesicle-like nanoparticles loaded with PDRN in different solutions, indicating that the structure of tea vesicle-like nanoparticles remains stable in different solutions and can be used for oral treatment of related diseases. Figure 8 As shown in the figure, the polydispersity index of tea vesicle-like nanoparticles loaded with PDRN in different solutions is less than 0.2, and the colloidal stability is better than that of tea vesicle-like nanoparticles, which is more suitable for oral application. Fig. 9 As shown, the TEM image of tea vesicle-like nanoparticles loaded with PDRN after incubation in gastrointestinal fluid for 2 hours. The tea vesicle-like nanoparticles loaded with PDRN maintained a uniform spherical structure with stable structure after incubation in gastrointestinal fluid for 2 hours.

[0066] Example 4

[0067] Tea vesicle-like nanoparticles loaded with PDRN are used orally to treat inflammatory bowel diseases, such as Fig.10 shown.

[0068] ①25 8-week-old male C57BL / 6J mice were raised in the Experimental Animal Center of the Medical Research Institute of Northwestern Polytechnical University. After one week of free diet adaptive feeding of mice, five mice were randomly selected as a normal group (blank group Control), and the other experimental mice were randomly divided into four groups: DSS modeling group (DSS modeling refers to the use of dextran sulfate sodium salt (DSS) to construct ulcerative colitis), PDRN group, EV group and PDRN-EV group. These four groups of mice were free to drink 2% to 3% DSS to establish an ulcerative colitis mouse model, and each mouse was marked and administered on day 0. Check the status of the mice and check the leakage of the water bottle every day.

[0069] ② During the entire experiment, the weight of each mouse was recorded to calculate the percentage of weight loss, and the feces of the mice were collected to reflect the diarrhea and bloody stools. Fig.11As shown, the weight of each mouse was recorded during the entire experiment to calculate the percentage of weight loss of the mouse. During the entire treatment period, the weight loss of the tea vesicle-like nanoparticles loaded with PDRN was about 3%, while the negative control DSS group was 18%. This shows that the tea vesicle-like nanoparticles loaded with PDRN can slow down the weight loss of colitis mice and alleviate the symptoms of colitis mice. Fig.12 As shown, tea vesicle-like nanoparticles loaded with PDRN can slow down the increase of disease activity index in colitis mice and relieve the symptoms of colitis mice. The sum of the scores of weight loss percentage, diarrhea score, and blood in stool score is used to calculate the disease activity index (DAI), which is the DAI value. That is, DAI = (body weight index + stool shape + bleeding). DAI scoring standard: symptom scoring once a day: 0-1% weight loss percentage is 0 points, 1%-5% is 1 point, 5%-10% is 2 points, 10%-20% is 3 points, and ≥20% is 4 points; no abnormal stool characteristics are 0 points, loose stools are 1 points, unformed stools are 2 points, and watery stools are 3 points; normal stools without blood are 0 points, stools with blood are 1 point, visible to the naked eye are 2 points, and heavy bleeding is 3 points. On the eighth day, the mice were killed by cervical dislocation, and the entire colon segment was dissected out and rinsed with saline. The length of the colon was measured and photographed, and the inflammatory condition of the mice was characterized by the length of the colon.

[0070] like Fig.13 As shown in the figure, the colon length of the normal control group was about 8 cm, and the colon length of the negative control DSS group was about 6.5 cm, which was significantly shortened, while the colon length of the tea vesicle-like nanoparticle group loaded with PDRN was about 7.8 cm, which indicates that the tea vesicle-like nanoparticles loaded with PDRN can inhibit the shortening of the colon of colitis mice and alleviate the symptoms of colitis mice. Fig.14 As shown, the blue arrow represents inflammatory cell infiltration; the red arrow represents mucosal epithelial erosion; the green arrow represents submucosal water. Tea vesicle-like nanoparticles loaded with PDRN significantly reduced DSS-induced colon damage and infiltration of inflammatory cells in the mucosal lamina propria, and effectively alleviated the severity of colitis compared with other treatment groups.

[0071] Therefore, the present invention adopts the above-mentioned tea vesicle-like nanoparticles loaded with PDRN, preparation method and application, and the tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides have a stable lipid bilayer structure in gastrointestinal simulated fluid and can be orally used to treat diseases. It provides a new strategy for the oral application of PDRN and has a good application prospect.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing tea vesicle-like nanoparticles loaded with PDRN, characterized in that: The steps include: Step 1, extracting tea vesicle-like nanoparticles, washing and chopping tea leaves to squeeze juice, collecting the initial product in a centrifuge bottle, performing differential centrifugation, discarding the precipitate after centrifugation, taking the supernatant, adding the supernatant to an ultracentrifuge and centrifuging to obtain tea vesicle-like nanoparticles; Step 2, preparing tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides, mixing polydeoxyribonucleotides with tea vesicle-like nanoparticles, incubating with low-power ultrasound at 37° C., 0-100 rpm, for 1-3 hours to promote membrane fusion; adding the mixed solution to an ultracentrifuge for centrifugation to remove free polydeoxyribonucleotides; Step three, characterize the tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides, determine the encapsulation efficiency and drug loading of polydeoxyribonucleotides in the tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides by an indirect method; use a dynamic light scattering particle size analyzer to determine the hydrodynamic particle size and polydispersity index of the tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides; use a nanoparticle tracking analyzer to detect the particle size of the tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides; and observe the structure of the tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides by transmission electron microscopy.

2. The method for preparing the tea vesicle-like nanoparticles loaded with PDRN according to claim 1, wherein: In step 1, the centrifugation parameters of the differential centrifugation are 1000×g for 10 min, 3000×g for 20 min, and 10000×g for 40 min.

3. The preparation method of tea vesicle-like nanoparticles loaded with PDRN according to claim 1, characterized in that: The centrifugation parameters in the ultracentrifuge are 130,000×g to 170,000×g for 60 to 90 min.

4. The preparation method of the tea vesicle-like nanoparticles of the PDRN load according to claim 1, characterized in that, In step 1, the specific operation of extracting tea vesicle-like nanoparticles is as follows: take 100g to 200g of tea, remove the thick stems and yellow leaves and insect leaves, wash them with ultrapure water for 1 to 3 times, chop them and pour them into a juicer, add 200mL to 400mL of ultrapure water, and squeeze the juice for 60 to 120S; squeeze the squeezed mixture with a squeezer, and collect the primary product in a centrifugal bottle; The primary product is balanced and placed in a high-speed centrifuge for differential centrifugation to remove large particles, dead cells and cell debris. The centrifugation parameters are 1000×g for 10 minutes, 3000×g for 20 minutes, and 10000×g for 40 minutes. After high-speed centrifugation, the precipitate is discarded, the supernatant is taken, and the supernatant is added to an ultracentrifuge, and centrifuged at 130000×g to 170000×g for 60 to 90 minutes to obtain tea extracellular vesicles, which are resuspended and collected using a 0.9% sodium chloride solution, and stored at -80°C to avoid repeated freezing and thawing when used.

5. The preparation method of the tea vesicle-like nanoparticles of the PDRN load according to claim 1, characterized in that, In step 2, the specific operation of preparing tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides is as follows: polydeoxyribonucleotides and tea vesicle-like nanoparticles are mixed in a ratio of 1 to 8:1, low-power ultrasound is applied, on for 10 to 30 seconds, off for 30 to 50 seconds, and 4 to 8 cycles of drug loading; incubation at 37°C, 0 to 100 rpm, for 1 to 3 hours to promote membrane fusion; the solution is added to an ultracentrifuge, and centrifuged at 130,000×g to 170,000×g for 60 to 90 minutes to remove free polydeoxyribonucleotides.

6. The preparation method of the tea vesicle-like nanoparticles loaded with PDRN according to claim 1, characterized in that, In step three, the indirect method for determining the encapsulation efficiency and drug loading of polydeoxyribonucleotides in tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides is as follows: 200 μL to 500 μL of drug-loaded tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides are added to a 100 kD to 300 kD ultrafiltration centrifuge tube, centrifuged at 10,000 rpm to 15,000 rpm for 10 min to 30 min, and the absorbance of the filtrate at 260 nm is detected.

7. The preparation method of the tea vesicle-like nanoparticles of the PDRN load according to claim 1, characterized in that, In step three, the operation of using a dynamic light scattering particle size analyzer to determine the flow dynamics particle size and polydispersity index of tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides is as follows: take an appropriate amount of tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides and dissolve them in 1.5-2 mL of deionized water, and the final concentration of the tea vesicle-like nanoparticles is 0.5-2 mg / mL. Use a dynamic light scattering particle size analyzer to determine the flow dynamics particle size and polydispersity index of the tea vesicle-like nanoparticles, and measure three times to obtain the average value.

8. The preparation method of tea vesicle-like nanoparticles loaded with PDRN according to claim 1, characterized in that, In step three, the operation of observing the structure of tea vesicle-like nanoparticles loaded with polydeoxyribonucleotides by transmission electron microscopy is as follows: take the mixed sample and drop it on the carbon support film copper mesh, and let it stand for 5 minutes; use filter paper to absorb the residual liquid from the edge of the copper mesh, and drop 2% phosphotungstic acid on the copper mesh, dye it at room temperature for 30 seconds, and use filter paper to absorb the excess dye. After naturally drying, place it under a transmission electron microscope for observation.

9. Tea vesicle-like nanoparticles loaded with PDRN, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the PDRN-loaded tea vesicle-like nanoparticles as claimed in claim 9 in the preparation of oral medications for treating inflammatory bowel disease.

Images