PDRN, supramolecular liposome containing the PDRN, and preparation method and use thereof

Abstract

The present invention discloses a novel PDRN, a supramolecular liposome containing the PDRN, and a preparation method and use thereof. The preparation method of the novel PDRN comprises: (1) mixing tissue raw materials with PBS, crushing, enzymolysis, adding SDS, Tween20 solution, polyethylene glycol octylphenyl ether, TE buffer, NaCl, stirring, centrifuging, mixing the supernatant with anhydrous ethanol, and standing to obtain a crude DNA extract; (2) using an ultrasonic interrupter to interrupt and shear the tissue; (3) using restriction endonucleases and deoxyribonucleases to perform enzymolysis, etc. The PDRN supramolecular liposome is obtained by encapsulating the above-mentioned PDRN with fatty acids extracted from marine microalgae. The present invention uses fish testis as a raw material source, broadens the range of raw material selection, reduces preparation costs, and the organic reagent used in the preparation method of the present invention is ethanol, which is easy to remove and relatively harmless.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a PDRN, a supramolecular liposome containing the PDRN, and a preparation method and use thereof. Background Art

[0002] Polydeoxyribonucleotides (PDRN) are a class of DNA derivatives that have a variety of pharmacological activities, such as tissue repair, anti-inflammation, wound healing, angiogenesis stimulation, and anti-ischemic damage. They are widely used in new therapies and technologies, as well as in the development of artificial biological replacement systems, to support, repair or replace damaged cells, tissues and organs. Currently, PDRN is mainly extracted from salmon ( Oncorhynchus keta ) or rainbow trout ( Oncorhynchus mykiss ) is extracted and purified from sperm cells of humans. The source of raw materials is limited and the preparation cost is high. At the same time, traditional methods of extracting and purifying PDRN often use phenol and chloroform, which are harmful to the human body, and the extraction steps are cumbersome and time-consuming.

[0003] Some people have also tried to produce PDRN from more readily available and lower-cost sources, such as fish testes. However, it is difficult to obtain PDRN of higher purity using fish testes as raw materials, especially the presence of lipids (a general term for oils, fats, and lipids), which will interfere with the extraction of PDRN and reduce the purity of the extracted PDRN, thereby reducing its efficacy and affecting its long-term preservation.

[0004] In addition, the prior art has the technology of making PDRN into supramolecular liposomes for use. Supramolecular liposomes are nano-sized particles formed by self-assembly of lipid molecules with unique biocompatibility and drug delivery capabilities. Supramolecular liposomes can optimize the release and absorption characteristics of drugs by adjusting their composition and structure, thereby improving the efficacy of drugs and reducing side effects. This technology has a wide range of application prospects in drug research and development and clinical applications. However, in the supramolecular liposome preparation process, the presence of lipids in PDRN affects the charge interaction between lipid molecules and PDRN, affecting embedding rate and particle size. Summary of the Invention

[0005] The main purpose of the present invention is to provide a PDRN using fish testis as raw material, with a simple process and a low lipid content in the product, a supramolecular liposome containing the PDRN, and a preparation method and use thereof.

[0006] To achieve the above object, the present invention provides a method for preparing PDRN, comprising the following steps:

[0007] (1) DNA extraction

[0008] The fish testis is beaten into a slurry to obtain a tissue material, the tissue material is mixed with PBS, and the slurry is disrupted using an ultrasonic cell disruptor. Papain is then added for enzymatic hydrolysis, and SDS, a lipid remover, and NaCl are then added to obtain a tissue mixture. The tissue mixture is stirred and centrifuged, and the supernatant is collected. The supernatant is then mixed with anhydrous ethanol and allowed to stand. Finally, flocculent DNA chromatographically separated from the upper layer is removed, washed, and dissolved in PBS to obtain a crude DNA extract.

[0009] (2) Cutting and shearing DNA

[0010] The crude DNA extract was placed in a centrifuge tube and sheared using an ultrasonic shearer to obtain DNA fragments;

[0011] (3) Preparation of PDRN by enzymatic hydrolysis

[0012] Restriction endonucleases and deoxyribonucleases are added to the broken DNA fragments for enzymatic hydrolysis, followed by centrifugation. The supernatant is collected and mixed with anhydrous ethanol, allowed to stand, and finally the flocculent DNA from the upper layer is taken out and washed to obtain PDRN.

[0013] Furthermore, in step (1), PBS is prepared by diluting 10×PBS buffer ten-fold, and the ratio of tissue material to PBS is 1 mg:10 mL.

[0014] Furthermore, in step (1), the working conditions of the crushing process are: working time 3 seconds, working interval 5 seconds, working power 90W, total working time 30 minutes, and 6mm amplitude rod.

[0015] Furthermore, in step (1), the amount of papain used is 10-12% of the weight of the tissue material, and the specific process of enzymatic hydrolysis is enzymatic hydrolysis in a constant temperature water bath at 37-40°C for 0.5-2 hours.

[0016] Furthermore, in step (1), the lipid remover consists of Tween 20, polyethylene glycol octylphenyl ether and TE buffer, the amount of Tween 20 is 0.1 wt%, the amount of polyethylene glycol octylphenyl ether is 0.2 ml / L, and the amount of TE buffer is 0.5 ml / L.

[0017] Furthermore, in step (1), the amount of SDS used is 10% of the weight of the tissue material, the amount of NaCl used is such that the final concentration in the tissue mixture reaches 1.3 to 1.6 mol / L, and the specific process of the stirring treatment is stirring in a constant temperature water bath at 37°C for 1 hour.

[0018] Furthermore, in step (2), the working conditions of the shearing treatment are: DNA concentration 3000 ng / µl, working time 3 seconds, working interval 5 seconds, working power 90 W, total working time 2 minutes, and 6 mm amplitude rod.

[0019] Furthermore, in step (3), the restriction endonuclease is selected from any one of Asp7181, Bcul, Not1, Xbal, Hindll1, and Kpn1, the deoxyribonuclease is DNaseI, the dosage of the restriction endonuclease and the deoxyribonuclease is 500 U / ml, and the specific process of the enzymatic hydrolysis is enzymatic hydrolysis in a constant temperature water bath at 37°C for 1 to 2 hours. More preferably, the restriction endonuclease Asp7181 is selected.

[0020] Furthermore, in steps (1) and (3), the centrifugal treatment conditions are both at a rotation speed of 10,000 rpm and a time of 15 minutes.

[0021] The present invention also provides a PDRN, which is prepared according to the above-mentioned preparation method.

[0022] The present invention also provides a method for preparing PDRN supramolecular liposomes, comprising the following steps:

[0023] (1) Dissolving fatty acids extracted from marine microalgae in ethanol to obtain a lipid-ethanol solution;

[0024] (2) dissolving the above-mentioned PDRN in citric acid buffer to obtain PDRN-citric acid buffer;

[0025] (3) The PDRN-citrate buffer solution and the lipid-ethanol solution are used as the inner phase and the outer phase, respectively, and loaded into a lipid nanoparticle synthesizer to synthesize liposomes to obtain the PDRN supramolecular liposomes.

[0026] Furthermore, the concentration of the lipid-ethanol solution was 7.5 mg / ml, the concentration of the PDRN-citrate buffer was 0.1 μg / μL, and the synthesis conditions of the lipid nanoparticle synthesizer were: an internal phase and external phase flow rate ratio of 1:5, a total flow rate of 24 ml / min, a total product volume of 36 ml, a front waste liquid of 2 ml, and a rear waste liquid of 0.1 ml.

[0027] The present invention also provides a PDRN supramolecular liposome, which is prepared according to the above preparation method.

[0028] The present invention also provides a use of the above-mentioned PDRN or the above-mentioned PDRN supramolecular liposome in the preparation of anti-inflammatory drugs or anti-allergic and antipruritic drugs.

[0029] The beneficial effects of the present invention are embodied in:

[0030] The invention adopts fish testis as a raw material source, broadens the range of raw material selection, reduces the preparation cost, and the organic reagent used in the preparation method of the invention is ethanol, which is easy to remove and relatively harmless.

[0031] In addition to polydeoxyribonucleotides and proteins, fish testicular tissue also contains a large amount of lipids, which affect the concentration and quality of extracted nucleic acids. Therefore, during the extraction process, the present invention has specially developed a lipid remover composed of Tween 20, polyethylene glycol octylphenyl ether, and TE buffer. This lipid remover has excellent emulsification ability and can effectively disperse the oil phase in the aqueous phase to form a stable emulsion system. It also has low toxicity, good biocompatibility, and is easy to remove. It has been proven to be able to effectively eliminate the effects of lipids.

[0032] During the DNA extraction process, the present invention uses an ultrasonic cell disruptor to break the cell membrane structure in the fish testis so that the deoxyribonucleic acid therein can be released in large quantities. The long fragments of deoxyribonucleic acid that have been extracted are broken into small fragments that are easier to function by an ultrasonic interrupter, that is, high-purity small molecule PDRN is obtained. High-frequency ultrasonic vibration can accurately break the phosphodiester bond of the DNA molecule through a short and intense sound wave impact, thereby achieving precise cutting of the DNA sequence. Compared with traditional physical or chemical methods, the ultrasonic interrupter can complete DNA fragmentation in a shorter time, and the size and distribution of the fragments are more uniform.

[0033] The present invention has discovered that restriction endonucleases and deoxyribonucleases can further reduce the fragment size of PDRN and ensure that the resulting PDRN fragments are undegraded, high-quality polydeoxyribonucleotides. In particular, the combined enzymatic digestion of restriction endonucleases Asp7181 and deoxyribonucleases produces the smallest PDRN fragments and the highest nucleic acid concentration, making it the most suitable enzymatic digestion step for PDRN.

[0034] The present invention utilizes the principle that the solubility of DNA in a sodium chloride solution changes with the concentration of sodium chloride. When the molar concentration of sodium chloride is a certain value, the solubility of DNA is low and proteins can be dissolved in the solution. By utilizing this principle, the DNA dissolved in the sodium chloride solution can be precipitated and most of the dissolved proteins can be removed. The present invention also utilizes the fact that DNA is insoluble in an alcohol solution, but certain substances in cells can be dissolved in an alcohol solution. By utilizing this principle, DNA containing fewer impurities can be further extracted.

[0035] The present invention uses fatty acids extracted from marine microalgae as carriers. Under acidic conditions, the extracted fatty acids can be protonated to form cationic lipids. These fatty acids bind to negatively charged PDRN through electrostatic interaction to form PDRN-loaded lipid nanoparticles, i.e., supramolecular liposomes. The prepared lipid nanoparticles have a high embedding efficiency and a small particle size, making them more easily absorbed through the skin and thus more effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is the concentration of DNA extracted at each level of the four factors in Experimental Example 2;

[0037] Figure 2 is the extraction rate of DNA extracted at each level of the four factors in Experimental Example 2;

[0038] Figure 3 is the expression level of TNF-α in dermal papilla cells in Experimental Example 6;

[0039] Figure 4 is the IL-6 expression level of dermal papilla cells in Experimental Example 6;

[0040] Figure 5 is the expression level of IL-1ß in dermal papilla cells in Experimental Example 6;

[0041] Figure 6 This is the wind group experiment effect diagram of Experimental Example 7. DETAILED DESCRIPTION

[0042] In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the following examples are given for illustration. It should be noted that the following examples do not limit the scope of protection claimed by the present invention.

[0043] Unless otherwise specified, the raw materials, reagents, and apparatus used in the following examples can be obtained from conventional commercial sources or by known methods. Unless otherwise specified, the methods used in the examples are all within the skill of those skilled in the art. The ultrasonic cell disruptor used was the SM-900D (Shunma, Nanjing), with a maximum power of 900W; the non-contact ultrasonic disruptor used was the XM-26A (Xiaomei Ultrasonics, Kunshan), with a maximum power of 900W; and the lipid nanoparticle synthesizer model was the LNP-S1-L (FluidicLab, Shanghai). Marine microalgae-derived fatty acids were purchased from INNOVA BAY (model SEL Schizochytrium sp. INNOVA23, Shenzhen, China). The carp testes were from the South China species.

[0044] Example 1

[0045] Preparation of PDRN

[0046] The specific steps are as follows:

[0047] (1) DNA extraction

[0048] The carp testes stored at -20°C were thawed in a 37°C water bath for 30 min, washed with deionized water, and then homogenized into a slurry to serve as the tissue material for DNA extraction.

[0049] 4 g of tissue material was taken and added to a 50 mL centrifuge tube with PBS (10× PBS buffer diluted ten times) at a weight-to-volume ratio of 1 mg:10 mL. The cells were disrupted using an ultrasonic cell disruptor under the following conditions: working time 3 seconds, working interval 5 seconds, working power 90 W, total working time 30 minutes, and 6 mm amplitude. After the disruption, 0.4 mg of papain was added to the centrifuge tube and enzymatic hydrolysis was carried out in a constant temperature water bath at 37°C for 1.5 hours. After the enzymatic hydrolysis was completed, 0.4 mg of SDS (sodium dodecyl sulfate) and 0.1 wt% of Tween 20, 0.2 ml / L of polyethylene glycol octylphenyl ether, and 0.5 ml / L of 1× TE buffer (pH 7.0) were added. 8.0), and added NaCl at a final concentration of 1.6 mol / L. The resulting tissue mixture was stirred at 200 rpm in a constant temperature water bath at 37°C for 1 hour. The tissue mixture was then placed in a high-speed centrifuge and centrifuged at 10,000 rpm for 15 minutes. The supernatant was collected and mixed with anhydrous ethanol (at a ratio of 2.5:1). After standing for 10 minutes, the flocculent DNA precipitated on the upper layer was removed and washed three times with 75% ethanol. Finally, the washed DNA precipitate was placed in a 50 mL centrifuge tube and fully dissolved in 40 mL of 1× PBS buffer. This was recorded as a crude DNA extract. Due to its high concentration, the crude extract was in a milky white gel-like state.

[0050] (2) Cutting and shearing DNA

[0051] The concentration of the crude DNA extract obtained in step (1) was measured using Nanodrop and calibrated to 3000 ng / µL. 7 mL of the solution was placed in a 50 mL centrifuge tube and sheared using a non-contact ultrasonic shearing device. The treatment conditions were: DNA concentration 3000 ng / µL, working time 3 seconds, working interval 5 seconds, working power 90 W, total working time 2 minutes, 6 mm amplitude rod. The mixture after treatment was recorded as DNA fragments. The DNA fragment with the highest abundance was 100 to 250 bp.

[0052] (3) Preparation of PDRN by enzymatic hydrolysis

[0053] Restriction endonuclease Asp7181 and deoxyribonuclease (DNaseI, Thermo Fisher) were added to the DNA fragments obtained in step (2) at a dosage of 500 U per ml of DNA fragments, and the fragments were enzymatically hydrolyzed in a constant temperature water bath at 37°C for 1.5 hours. The enzymatically hydrolyzed solution was then transferred to a new 50 ml centrifuge tube, placed in a high-speed centrifuge, and centrifuged at 10,000 rpm for 15 minutes. The supernatant was collected and mixed with anhydrous ethanol (at a ratio of 2.5:1). After standing for 10 minutes, the flocculent DNA precipitated on the upper layer was removed and washed three times with 75% ethanol to obtain PDRN.

[0054] Experimental Example 1

[0055] Effect of lipid removal agents on the purity of PDRN

[0056] Example 1 was used as the experimental group. On the basis of Example 1, only the lipid remover was omitted or the components of the lipid remover were adjusted (equal replacement) as the control group. Then, the total lipid content of the PDRN extracted from the experimental group and the control group was detected using a colorimetric quantitative detection kit. The results are shown in Table 1.

[0057] Table 1

[0058]

[0059] Note: + indicates that the component is added, - indicates that the component is not added.

[0060] The results showed that when Tween 20, polyethylene glycol octylphenyl ether, and TE buffer were used as lipid removers, the residual heavy lipid content of PDRN extracted was the lowest, at 19 mg / dL. When no lipid remover was added, the residual heavy lipid content of PDRN extracted reached 346 mg / dL, which was 18.37 times that of the group with added lipid removers. This shows that the present invention can effectively reduce the lipid content of PDRN extracted by using Tween 20, polyethylene glycol octylphenyl ether, and TE buffer as lipid removers, and the degreasing effect of this combination is better than all other control groups.

[0061] Experimental Example 2

[0062] Effects of enzymatic hydrolysis temperature, final salt concentration, enzyme dosage, and enzymatic hydrolysis time on DNA extraction efficiency

[0063] In order to explore the effects of enzymatic hydrolysis temperature, final salt (NaCl) concentration, amount of enzyme added, and enzymatic hydrolysis time on the DNA extraction rate in step (1), single-factor experiments were performed according to the method of Example 1. The amount of enzyme added was calculated based on the weight of the tissue added, and the experimental design is shown in Table 2.

[0064] Table 2 Single-factor experimental design for DNA extraction

[0065]

[0066] DNA extraction rate = (cV / m) × 100%, where c is the concentration of the crude DNA extract, V is 1 mL of 1× PBS buffer used to dissolve the DNA precipitate, and m is 0.1 g of fish testis tissue.

[0067] The results of the single-factor experiment are as follows Figure 1 and Figure 2 As shown in Table 3, the response surface optimization conditions were set according to the results. According to the Box-Behnken (BBD) experimental design, the optimal response surface prediction scheme was obtained as shown in Table 3.

[0068] Table 3 Response surface prediction optimal solution

[0069]

[0070] The experimental conditions in Table 3 were used to conduct the actual value detection experiment. The tissue dosage was 4 g per experimental group, and three parallel groups were set up. The final DNA solution was diluted to 20,000 μL. Using the formula: (DNA concentration × DNA solution volume) / tissue amount, the actual yield was calculated to be 0.74%, while the predicted yield was 0.85%. This indicates that the actual value is relatively small compared to the predicted value. The standard deviation between the two values was calculated to be 0.003 < 0.05, indicating that the difference between the predicted and actual values is significantly small. This also shows that the predicted value is achievable in practice and that the optimal experimental protocol can be used as the optimal DNA extraction protocol.

[0071] Experimental Example 3

[0072] Effects of ultrasonic shearing conditions on DNA fragmentation

[0073] In order to explore the effects of factors such as DNA concentration, total working time, and fragmentation power on the DNA fragmentation effect in step (2), single-factor experiments were performed according to the method of Example 1, and the settings of each factor and each level are shown in Table 4 below.

[0074] Table 4 Single-factor experimental design of DNA fragmentation

[0075]

[0076] The test results are shown in Tables 5 to 7:

[0077] Table 5 Band distribution and abundance range of the gradient over the total working time

[0078]

[0079] Table 6 Band distribution and abundance range of working power gradient

[0080]

[0081] Table 7 Band distribution and abundance range of sample concentration gradient

[0082]

[0083] Experimental Example 4

[0084] Effect of enzyme selection on PDRN size

[0085] To explore the effect of enzyme type on the size of the prepared PDRN fragments, according to the method of Example 1, a variety of restriction endonucleases were used alone or in combination with deoxyribonuclease DNaseI to enzymatically digest the DNA fragments, and then the fragment size and concentration were measured by agarose gel electrophoresis and Nanopdrop. The results are shown in Table 8.

[0086] Table 8 Effects of endonuclease types and deoxyribonucleases on PDRN fragment size and concentration

[0087]

[0088] Note: + indicates that the component is added, - indicates that the component is not added.

[0089] The results showed that under the co-enzymatic hydrolysis of restriction endonuclease Asp7181 and deoxyribonuclease DNaseI, the obtained PDRN fragment was the smallest and the nucleic acid concentration was higher, which was the most suitable enzymatic hydrolysis step for PDRN.

[0090] Example 2

[0091] Preparation of PDRN supramolecular liposomes

[0092] The specific steps are as follows:

[0093] (1) Preparation of lipid-ethanol solution

[0094] Using ethanol as a solvent, the fatty acids extracted from marine microalgae were dissolved in ethanol to prepare a lipid-ethanol solution with a concentration of 7.5 mg / ml;

[0095] (2) Preparation of PDRN-citrate buffer

[0096] Prepare 50 ml of 100 mM citric acid monohydrate solution and 50 ml of sodium citrate dihydrate solution using ultrapure water. Take 33.0 ml of citric acid solution and 17.0 ml of sodium citrate solution, mix them, add DEPC, let them stand for 30 minutes, and then sterilize them by autoclave to remove DEPC. After sterilization, dilute the volume to 100 ml with DEPC water to obtain 50 mM citric acid buffer with a pH of 4.

[0097] Using the above-mentioned citric acid buffer as a solvent, the PDRN prepared in Example 1 was dissolved in the citric acid buffer to prepare a PDRN-citric acid buffer with a concentration of 0.1 μg / μL;

[0098] (3) The PDRN-citrate buffer solution and lipid-ethanol solution were filtered through a 0.22 μm filter membrane, and then loaded into a lipid nanoparticle synthesizer as the inner and outer phases for liposome synthesis. The synthesis conditions were: the flow rate ratio of the inner and outer phases was 1:5, the total flow rate was 24 ml / min, the total volume of the product was 36 ml, the front waste liquid was 2 ml, and the rear waste liquid was 0.1 ml. The outflowing liquid was collected with a collection tube to obtain PDRN supramolecular liposomes.

[0099] Experimental Example 5

[0100] Determination of the encapsulation efficiency and particle size of PDRN supramolecular liposomes

[0101] Encapsulation efficiency determination

[0102] Principle: Quant-iT™ RiboGreen® nucleic acid reagent is an ultrasensitive fluorescent nucleic acid stain capable of detecting 1-200 ng of nucleic acids in solution. This nucleic acid dye is impermeable to LNPs, so only free nucleic acids not encapsulated by the LNPs can be bound. Triton-100, a surfactant commonly used as a demulsifier, is used to treat the LNP-PDRN obtained with 1% Triton-100, releasing the encapsulated nucleic acids and determining the total nucleic acid content. The drug loading is calculated by dividing the difference in nucleic acid content before and after demulsification by the total nucleic acid content to determine the encapsulation efficiency: Encapsulation efficiency (%) = (post-demulsification quantity - pre-demulsification quantity) / post-demulsification quantity.

[0103] Experimental Method Kit: Quant-iT™ PicoGreen™ dsDNA Assay Kit. Fluorescence intensity was read using SPARK with excitation at 480 nm and emission at 520 nm.

[0104] Drug loading = reading after demulsification - reading before demulsification.

[0105] Encapsulation efficiency (%) = drug loading / reading after demulsification.

[0106] The PDRN supramolecular liposomes prepared in Example 2 and the preparation method of Example 2 were used to prepare PDRN supramolecular liposomes using different encapsulating lipids and different solvents (instead of citric acid buffer), and the encapsulation efficiency was measured. The results are shown in Table 9:

[0107] Table 9 Effects of different solvents and encapsulating lipids on PDRN encapsulation efficiency

[0108]

[0109] Particle size determination:

[0110] The particle size of different PDRN supramolecular liposomes was determined using a laser particle size analyzer as follows: The laser particle size analyzer used was Bettersize 2600 (Dandong Bettersize, Liaoning). 100 mg of the sample to be tested was added to the test water tank, and the water inlet button was clicked to fill the test water tank in the instrument with the resuspension. The circulation speed was set to 120 rpm. After vortexing for 2 minutes, the sample to be tested was fully resuspended in the test water tank and defoamed. The ultrasonic power was set to 20%, the background value was 1.2, the detection interval was 3 seconds, and the test was repeated three times to complete the PDRN supramolecular liposome particle size detection. The results are shown in Table 10:

[0111] Table 10 Effects of different solvents and encapsulating lipids on PDRN particle size

[0112]

[0113] Experimental Example 6

[0114] Anti-inflammatory effect test of PDRN supramolecular liposomes

[0115] The fish testis-based PDRN prepared in Example 1, the PDRN supramolecular liposomes prepared in Example 2, and the PDRN supramolecular liposomes prepared according to the preparation method of Example 2 using different encapsulating lipids were co-incubated with lipopolysaccharide (LPS) and DPC to verify their anti-inflammatory effects on cells.

[0116] Cell culture: After culturing DPC cells in a 24-well plate for 24 h, 1 μg / mL LPS solution was added to all wells except the blank group. Then, different concentrations of unencapsulated PDRN and encapsulated PDRN supramolecular liposomes were added and cultured at 37°C for 24 h.

[0117] Set up the groups as follows:

[0118] CK group: blank group;

[0119] LPS group: without PDRN and PDRN supramolecular liposomes;

[0120] Unwrapped-50 group: PDRN prepared in Example 1, concentration 500 mg / ml;

[0121] Unwrapped-100 group: PDRN prepared in Example 1, concentration 100 mg / ml;

[0122] Algae oil encapsulated-50 group: PDRN supramolecular liposomes prepared in Example 2, concentration 50 mg / ml;

[0123] Algae oil-encapsulated group-100: PDRN supramolecular liposomes prepared in Example 2, concentration 100 mg / ml;

[0124] Soybean lecithin-coated group-50: PDRN supramolecular liposomes were prepared according to the preparation method of Example 2, except that the fatty acids extracted from marine microalgae were replaced with soybean lecithin at a concentration of 50 mg / ml;

[0125] Soybean lecithin-encapsulated group-100: PDRN supramolecular liposomes were prepared according to the preparation method of Example 2, except that soybean lecithin was used instead of fatty acids extracted from marine microalgae to prepare PDRN supramolecular liposomes at a concentration of 100 mg / ml;

[0126] The results are as follows Figure 3-5 As shown in the figure, CK is the blank group. In the LPS-induced inflammatory response, PDRN can effectively inhibit the expression of inflammatory factors, among which PDRN encapsulated with fatty acids extracted from marine microalgae showed better anti-inflammatory effects.

[0127] Experimental Example 7

[0128] Wheal test

[0129] In the allergic wheal test, histamine is used as an allergen and applied to the skin. This causes symptoms such as redness, swelling, and itching, forming a wheal. The skin's reaction to the application of different groups of samples is then observed and measured. This wheal test allows the anti-allergic and antipruritic effects of the samples to be evaluated.

[0130] Set up the groups as follows:

[0131] CK group: sterile water;

[0132] Unencapsulated PDRN-100 group: PDRN prepared in Example 1, concentration 100 mg / ml;

[0133] Encapsulated PDRN-50 group: PDRN supramolecular liposomes prepared in Example 2, concentration 50 mg / ml;

[0134] Encapsulated PDRN-100 group: PDRN supramolecular liposomes prepared in Example 2, with a concentration of 100 mg / ml.

[0135] Experimental methods:

[0136] 1. First apply 200mg / ml histamine to the tested area. After 40-60 minutes, obvious redness, swelling and wheal will appear on the tested area, accompanied by itching symptoms.

[0137] 2. Add 25 μl of test sample solution of different concentrations into the spot tester

[0138] 3. Take photos and record any sensory sensations such as itching and tingling according to the test time.

[0139] See the results Figure 6 In the figure, a is before treatment, b is 1 hour after treatment, c is 2 hours after treatment, and d is 4 hours after treatment. The upper left, upper right, lower left, and lower right are respectively the CK group, the unwrapped PDRN-100 group, the wrapped PDRN-50 group, and the wrapped PDRN-100 group. The degree of redness, swelling, and itching is shown in Table 10:

[0140] Table 10 Redness, swelling and itching degree of wheal test

[0141]

[0142] For redness, swelling and itching, + indicates the degree, + indicates mild symptoms, ++ indicates moderate symptoms, +++ indicates severe symptoms, and - indicates no symptoms. For tolerability, - indicates that it is difficult to tolerate, - indicates that it is tolerable, - indicates that there is only a slight abnormal feeling, and + indicates no adverse feeling.

[0143] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. The preparation method of PDRN is characterized in that, The following steps are involved: (1) DNA extraction The fish testis is beaten into a slurry to obtain a tissue material, the tissue material is mixed with PBS, and the slurry is disrupted using an ultrasonic cell disruptor. Papain is then added for enzymatic hydrolysis, and SDS, a lipid remover, and NaCl are then added to obtain a tissue mixture. The tissue mixture is stirred and centrifuged, and the supernatant is collected. The supernatant is then mixed with anhydrous ethanol and allowed to stand. Finally, flocculent DNA precipitated in the upper layer is removed, washed, and dissolved in PBS to obtain a crude DNA extract. The lipid remover is composed of Tween 20, polyethylene glycol octylphenyl ether and TE buffer, the amount of Tween 20 is 0.1wt%, the amount of polyethylene glycol octylphenyl ether is 0.2ml / L, and the amount of TE buffer is 0.5ml / L; The dosage of papain is 10-12% of the weight of the tissue material, and the specific process of enzymatic hydrolysis is enzymatic hydrolysis in a constant temperature water bath at 37-40°C for 0.5-2 hours; (2) Cutting and shearing DNA The crude DNA extract was placed in a centrifuge tube and sheared using an ultrasonic shearer to obtain DNA fragments; The treatment conditions were: DNA concentration 3000 ng / µl, working time 3 s, working interval 5 s, working power 90 W, total working time 2 min, 6 mm horn; (3) Preparation of PDRN by enzymatic hydrolysis Restriction endonucleases and deoxyribonucleases are added to the broken DNA fragments for enzymatic digestion, followed by centrifugation. The supernatant is collected and mixed with anhydrous ethanol, allowed to stand, and finally the flocculent DNA from the upper layer is taken out and washed to obtain PDRN. Asp7181 was selected as restriction endonuclease, DNaseI was selected as deoxyribonuclease, and the dosage of restriction endonuclease and deoxyribonuclease was 500 U / ml. The specific process of enzymatic hydrolysis was enzymatic hydrolysis in a constant temperature water bath at 37° C. for 1 to 2 hours.

2. The preparation method of PDRN as claimed in claim 1, characterized in that, In step (1), the amount of SDS is 10% of the weight of the tissue material, the amount of NaCl is such that the final concentration in the tissue mixture reaches 1.3 to 1.6 mol / L, and the specific process of the stirring treatment is stirring in a constant temperature water bath at 37°C for 1 hour. 3.PDRN, characterized in that Prepared according to the preparation method as claimed in claim 1 or 2.

4. A method for preparing PDRN supramolecular liposomes, characterized in that, The following steps are involved: (1) Dissolving fatty acids extracted from marine microalgae in ethanol to obtain a lipid-ethanol solution; (2) dissolving the PDRN as claimed in claim 3 in a citric acid buffer solution to obtain a PDRN-citric acid buffer solution; (3) The PDRN-citrate buffer solution and the lipid-ethanol solution are used as the inner phase and the outer phase, respectively, and loaded into a lipid nanoparticle synthesizer to synthesize liposomes to obtain the PDRN supramolecular liposomes.

5. the preparation method of PDRN supramolecular liposome as claimed in claim 4, is characterized in that, The concentration of the lipid-ethanol solution was 7.5 mg / ml, the concentration of the PDRN-citrate buffer was 0.1 μg / μL, and the synthesis conditions of the lipid nanoparticle synthesizer were: the flow rate ratio of the inner phase to the outer phase was 1:5, the total flow rate was 24 ml / min, the total volume of the product was 36 ml, the front waste liquid was 2 ml, and the rear waste liquid was 0.1 ml.

6. A PDRN supramolecular liposome, characterized in that Prepared according to the preparation method as claimed in claim 4 or 5.

7. The use of PDRN as claimed in claim 3 or PDRN supramolecular liposomes as claimed in claim 6 in the preparation of anti-inflammatory drugs or anti-allergic and antipruritic drugs.

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