Novel PDRN, supramolecular liposome containing PDRN as well as preparation method and application of supramolecular liposome

Abstract

The invention discloses a novel PDRN, a supramolecular liposome containing the PDRN as well as a preparation method and application of the supramolecular liposome. The preparation method of the novel PDRN comprises the following steps: (1) mixing a tissue raw material and PBS, crushing, carrying out enzymolysis, adding SDS, a Tween20 solution, polyethylene glycol octylphenol ether, a TE buffer solution and NaCl, stirring, centrifuging, mixing a supernatant with absolute ethyl alcohol, and standing to obtain a DNA crude extract; (2) carrying out breaking shearing treatment by using an ultrasonic breaking instrument; and (3) carrying out enzymolysis treatment by using restriction endonuclease and deoxyribonuclease, and the like. The PDRN supramolecular liposome is obtained by wrapping the PDRN with fatty acid extracted from marine microalgae. The fish testis are used as the raw material source, the selection range of the raw materials is widened, the preparation cost is reduced, and the organic reagent used in the preparation method is ethyl alcohol which is easy to remove and harmless.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a novel 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. Due to their multiple pharmacological activities such as tissue repair, anti-inflammation, wound healing, angiogenesis stimulation, and anti-ischemic injury, they are widely used in new therapies and technologies, as well as the development of artificial biological replacement systems, to support, repair or replace damaged cells, tissues and organs. At present, PDRN is mainly extracted and purified from sperm cells of salmon (Oncorhynchus keta) or rainbow trout (Oncorhynchus mykiss). The source of raw materials is limited and the preparation cost is high. At the same time, the traditional method of extracting and purifying PDRN often uses 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 easily available and lower-cost sources, such as fish testicles. However, it is difficult to obtain PDRN with higher purity using fish testicles 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 development and clinical applications. However, in the process of preparing supramolecular liposomes, the presence of lipids in PDRN affects the charge interaction between lipid molecules and PDRN, affecting the embedding rate and particle size. Summary of the invention

[0005] The main purpose of the present invention is to provide a PDRN which uses fish testis as raw material, has 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 a novel 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 tissue material is crushed by an ultrasonic cell disruptor, and then papain is added for enzymatic hydrolysis, and then SDS, a lipid remover and NaCl are added to obtain a tissue mixture, the tissue mixture is stirred, and then centrifuged, and the supernatant is collected, and then the supernatant is mixed with anhydrous ethanol, and allowed to stand, and finally the flocculent DNA chromatographed from the upper layer is taken out, washed, and dissolved in PBS to obtain a crude DNA extract;

[0009] (2) Cutting and shearing DNA

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

[0011] (3) Enzymatic hydrolysis to prepare PDRN

[0012] Restriction endonucleases and deoxyribonucleases are added to the broken DNA fragments for enzymatic treatment, followed by centrifugation, and the supernatant is collected. The supernatant is mixed with anhydrous ethanol and allowed to stand. 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 is composed of Tween 20, polyethylene glycol octyl phenyl ether and TE buffer, the amount of Tween 20 is 0.1wt%, the amount of polyethylene glycol octyl phenyl ether is 0.2ml / L, and the amount of TE buffer is 0.5ml / L.

[0017] Furthermore, 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 1.3-1.6 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] Further, 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 500U / ml, and the specific process of the enzymatic treatment 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 novel 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 novel PDRN in a citric acid buffer to obtain a 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: inner and outer phase flow rate ratio of 1:5, total flow rate of 24 ml / min, total product volume of 36 ml, front waste liquid of 2 ml, and rear waste liquid of 0.1 ml.

[0027] The present invention also provides a PDRN supramolecular liposome, which is prepared according to the above-mentioned 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 the raw material source, broadens the raw material selection range, 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 Tween20, polyethylene glycol octylphenyl ether and TE buffer. The lipid remover has excellent emulsification ability, can effectively disperse the oil phase in the water phase to form a stable emulsion system, has low toxicity and good biocompatibility, is easy to remove, and has been verified to be able to effectively eliminate the influence of lipids.

[0032] In 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, and the extracted long fragments of deoxyribonucleic acid are broken into small fragments that are easier to function by an ultrasonic interrupter, that is, high-purity small molecule PDRN is obtained, and high-frequency ultrasonic vibration can accurately break the phosphodiester bond of the DNA molecule through a short and strong sound wave impact, thereby achieving accurate 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 found that restriction endonucleases and deoxyribonucleases can further reduce the fragment size of PDRN and ensure that the obtained PDRN fragments are undegraded high-quality polydeoxyribonucleotides. In particular, under the co-enzymatic hydrolysis of restriction endonucleases Asp7181 and deoxyribonucleases, the obtained PDRN fragments are the smallest and the nucleic acid concentration is also high, which is the most suitable enzymatic hydrolysis 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 protein 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 protein can be removed at the same time. The present invention 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 less impurities can be further extracted.

[0035] The marine microalgae extracted fatty acids of the present invention are used as carriers. The marine microalgae extracted fatty acids can be protonated under acidic conditions to form cationic lipids, which are combined with negatively charged PDRN through electrostatic action to form lipid nanoparticles loaded with PDRN, i.e. supramolecular liposomes. The prepared lipid nanoparticles have a high embedding rate and a small particle size, and are more easily absorbed by the human body through the skin, thereby better exerting their efficacy. 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 IL-1β expression level of 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 scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0043] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial channels or can be obtained by existing known methods; unless otherwise specified, the methods used in the embodiments of the present invention are methods known to those skilled in the art. Among them, the ultrasonic cell disruptor uses SM-900D (Shunma, Nanjing), with a maximum power of 900W; the non-contact ultrasonic interrupter uses XM-26A (Xiaomei Ultrasound, Kunshan), with a maximum power of 900W; the lipid nanoparticle synthesizer model is LNP-S1-L (FluidicLab, Shanghai); marine microalgae extracted fatty acids are purchased from INNOVA BAY (model SEL Schizochytrium sp. INNOVA23, Shenzhen, China); carp testis is the testis of the South China carp variety.

[0044] Example 1

[0045] Preparation of novel PDRN

[0046] The specific steps are as follows:

[0047] (1) DNA extraction

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

[0049] Take 4g tissue material, add the tissue material and PBS (10×PBS buffer diluted ten times) in a weight volume ratio of 1mg:10mL to a 50mL centrifuge tube, and use an ultrasonic cell disruptor to disrupt the cells. The processing conditions are: working time 3 seconds, working interval 5 seconds, working power 90W, total working time 30 minutes, 6mm amplitude rod. After the disruption, add 0.4mg of papain to the centrifuge tube, and perform enzymolysis in a 37°C constant temperature water bath for 1.5 hours. After the enzymolysis, add 0.4mg of SDS (sodium dodecyl sulfate) and 0.1wt% of Tween20, 0.2ml / L of polyethylene glycol octylphenyl ether and 0.5ml / L of 1×TE buffer (pH 5.0) accounting for 0.1wt% of the mixture after the enzymolysis. 8.0), and add NaCl with a final concentration of 1.6 mol / L, the obtained tissue mixture is stirred at 200 rpm in a 37°C constant temperature water bath for 1 hour, then the tissue mixture is placed in a high-speed centrifuge, centrifuged at 10000 rpm for 15 minutes, the supernatant is collected, the supernatant is mixed with anhydrous ethanol (the ratio is 2.5:1), and after standing for 10 minutes, the flocculent DNA chromatographically obtained on the upper layer is taken out, and washed repeatedly with 75% ethanol three times, and finally the washed DNA precipitate is placed in a 50 mL centrifuge tube, and 40 mL 1×PBS buffer is added to fully dissolve it, which is recorded as a crude DNA extract. Due to its high concentration, the crude extract is in a milky white gel state;

[0050] (2) Cutting and shearing DNA

[0051] The concentration of the crude DNA extract obtained in step (1) was measured by Nanodrop and calibrated to 3000 ng / μL. 7 mL of the solution was taken into a 50 mL centrifuge tube and sheared using a non-contact ultrasonic shearing instrument. 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 fragmentation. The DNA fragment with the highest abundance obtained was 100 to 250 bp.

[0052] (3) Enzymatic hydrolysis to prepare PDRN

[0053] Restriction endonuclease Asp7181 and deoxyribonuclease (DNaseI, Thermo Fisher) were added to the DNA fragments obtained in step (2) at an amount of 500U per ml of DNA fragments, respectively, and 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 50ml centrifuge tube, placed in a high-speed centrifuge, and centrifuged at 10,000rpm for 15 minutes. The supernatant was collected and mixed with anhydrous ethanol (in a ratio of 2.5:1). After standing for 10 minutes, the flocculent DNA chromatogramed from the upper layer was taken out and washed repeatedly with 75% ethanol three times to obtain PDRN.

[0054] Experimental Example 1

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

[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, and 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]

[0060] Note: + indicates that the component is added, - indicates that the component is not added.

[0061] The results showed that when Tween20, polyethylene glycol octyl phenyl ether and TE buffer were used as lipid removers, the residual heavy lipid content of PDRN extracted was the lowest, which was 19 mg / dL, while 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 lipid remover added. This shows that the present invention can effectively reduce the lipid content of PDRN extracted by using Tween20, polyethylene glycol octyl phenyl ether and TE buffer as lipid removers, and the degreasing effect of this combination is better than all other control groups.

[0062] Experimental Example 2

[0063] Effects of enzymatic hydrolysis temperature, final salt concentration, enzyme dosage, and enzymatic hydrolysis time on DNA extraction efficiency

[0064] In order to explore the effects of the 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 according to the weight of the added tissue, and the experimental design is shown in Table 2.

[0065] Table 2 Single factor experimental design for DNA extraction

[0066]

[0067] DNA extraction rate = (cV / m) × 100%, where c is the concentration of the crude DNA extract, V is 1 mL of 1×PBS buffer for dissolving the DNA precipitate, and m is 0.1 g of fish testicular tissue material.

[0068] 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 solution for response surface prediction was obtained as shown in Table 3.

[0069] Table 3 Optimal solution for response surface prediction

[0070]

[0071] The actual value detection experiment was carried out using the experimental conditions in Table 3. The amount of tissue used was 4 g per group of experiments. Three parallel groups were set up. The final DNA solution was fixed to 20,000 μL. The actual yield was calculated to be 0.74% and the predicted yield was 0.85% by the formula: (DNA concentration × DNA solution volume) / tissue amount. It can be seen that compared with the predicted value, the actual value is relatively small. The standard deviation of the two was calculated and the standard deviation was 0.003<0.05, indicating that the difference between the predicted value and the actual value is significantly small. This also shows that the predicted value can be achieved in reality, and the best experimental plan can be used as the best DNA extraction plan.

[0072] Experimental Example 3

[0073] Effects of ultrasonic shearing conditions on DNA fragmentation

[0074] 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.

[0075] Table 4 DNA fragmentation single factor experimental design

[0076]

[0077] The test results are shown in Tables 5 to 7:

[0078] Table 5 Band distribution and abundance range of the gradient of total working time

[0079]

[0080] Table 6 Band distribution and abundance range of working power gradient

[0081]

[0082] Table 7 Band distribution and abundance range of sample concentration gradient

[0083]

[0084] Experimental Example 4

[0085] Effect of enzyme selection on PDRN size

[0086] In order to explore the effect of the type of enzyme on the size of the prepared PDRN fragments, according to the method of Example 1, a variety of restriction endonucleases were used alone or together 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.

[0087] Table 8 Effects of endonuclease types and deoxyribonucleases on PDRN fragment size and concentration

[0088]

[0089]

[0090] Note: + indicates that the component is added, - indicates that the component is not added.

[0091] 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 also higher, which was the most suitable enzymatic hydrolysis step for PDRN.

[0092] Example 2

[0093] Preparation of PDRN supramolecular liposomes

[0094] The specific steps are as follows:

[0095] (1) Preparation of lipid-ethanol solution

[0096] 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;

[0097] (2) Preparation of PDRN-citrate buffer

[0098] Use ultrapure water to prepare 50 ml of 100 mM citric acid monohydrate solution and sodium citrate dihydrate solution, respectively. Take 33.0 ml of citric acid solution and 17.0 ml of sodium citrate solution, mix them, add DEPC, let stand for 30 minutes, and then sterilize by high pressure to remove DEPC. After sterilization, use DEPC water to make up to 100 ml to obtain 50 mM citric acid buffer solution with pH = 4.

[0099] 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;

[0100] (3) The PDRN-citrate buffer and lipid-ethanol solution were filtered through a 0.22 μm filter membrane, respectively, and then loaded into a lipid nanoparticle synthesizer as the inner and outer phases for liposome synthesis. The synthesis conditions were: an inner and outer phase flow rate ratio of 1:5, a total flow rate of 24 ml / min, a total product volume of 36 ml, 2 ml of the front waste liquid, and 0.1 ml of the rear waste liquid. The effluent was collected with a collection tube to obtain PDRN supramolecular liposomes.

[0101] Experimental Example 5

[0102] Determination of encapsulation efficiency and particle size of PDRN supramolecular liposomes

[0103] Encapsulation efficiency determination

[0104] Principle: Quant-iT TM Nucleic acid reagent is an ultra-sensitive fluorescent nucleic acid stain that can detect 1-200ng of nucleic acid in solution. This nucleic acid dye cannot penetrate LNP, so only free nucleic acid not encapsulated by LNP can be bound. Triton-100 is often used as a demulsifier as a surfactant. LNP-PDRN obtained by treating with 1% Triton-100 can release the encapsulated nucleic acid and obtain the total nucleic acid amount. The drug loading amount is obtained by calculating the difference in the amount of nucleic acid before and after demulsification, and then divided by the total amount of nucleic acid to obtain the encapsulation rate, that is: Encapsulation rate (%) = (quantity after demulsification - quantity before demulsification) / quantity after demulsification.

[0105] Experimental method Kit: Quant-iT TM PicoGreen TM dsDNA Assay Kit uses SPARK to read the fluorescence intensity, with the excitation light set to 480nm and the emission light set to 520nm.

[0106] Drug loading = reading after demulsification - reading before demulsification.

[0107] Encapsulation efficiency (%) = drug loading / reading after demulsification.

[0108] 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:

[0109] Table 9 Effects of different solvents and encapsulating lipids on PDRN encapsulation efficiency

[0110]

[0111] Particle size determination:

[0112] The method for determining the particle size of different PDRN supramolecular liposomes using a laser particle size analyzer is as follows: The laser particle size analyzer uses Bettersize2600 (Dandong Bettersize, Liaoning). Add 100 mg of the sample to be tested to the detection tank, and click the water inlet button to fill the detection tank in the instrument with the resuspension. Set the circulation speed to 120 rpm, vortex for 2 minutes, and fully resuspend the sample in the detection tank and defoam. Set the ultrasonic power to 20%, the background value to 1.2, the detection interval to 3s, repeat three times, and complete the PDRN supramolecular liposome particle size detection. The results are shown in Table 10:

[0113] Table 10 Effects of different solvents and encapsulated lipids on PDRN particle size

[0114]

[0115] Experimental Example 6

[0116] Test of the anti-inflammatory effect of PDRN supramolecular liposomes

[0117] 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.

[0118] 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, and then different concentrations of unencapsulated PDRN and encapsulated PDRN supramolecular liposomes were added, and cultured at 37°C for 24 h.

[0119] Set up the groups as follows:

[0120] CK group: blank group;

[0121] LPS group: without PDRN and PDRN supramolecular liposomes;

[0122] Unwrapped-50 group: PDRN prepared in Example 1, concentration 500 mg / ml;

[0123] Unwrapped-100 group: PDRN prepared in Example 1, concentration 100 mg / ml;

[0124] Algae oil encapsulation-50 group: PDRN supramolecular liposomes prepared in Example 2, concentration 50 mg / ml;

[0125] Algae oil encapsulation-100 group: PDRN supramolecular liposomes prepared in Example 2, concentration 100 mg / ml;

[0126] Soybean lecithin encapsulated-50 group: According to the preparation method of Example 2, soybean lecithin was used to replace the fatty acids extracted from marine microalgae to prepare PDRN supramolecular liposomes with a concentration of 50 mg / ml;

[0127] Soybean lecithin encapsulated-100 group: According to the preparation method of Example 2, soybean lecithin was used to replace the fatty acids extracted from marine microalgae to prepare PDRN supramolecular liposomes with a concentration of 100 mg / ml;

[0128] 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 by fatty acids extracted from marine microalgae showed better anti-inflammatory effects.

[0129] Experimental Example 7

[0130] Wheal test

[0131] In the allergic wheal test, histamine is used as an allergen and applied to the skin, which will cause symptoms such as redness, swelling, and itching, forming a wheal. Then the skin reaction after applying different groups of samples is observed and measured. The wheal test can be used to evaluate the anti-allergic and antipruritic effects of the samples.

[0132] Set up the groups as follows:

[0133] CK group: sterile water;

[0134] Unencapsulated PDRN-100 group: PDRN prepared in Example 1, concentration 100 mg / ml;

[0135] PDRN-50 group after encapsulation: PDRN supramolecular liposomes prepared in Example 2, concentration 50 mg / ml;

[0136] PDRN-100 group after encapsulation: PDRN supramolecular liposomes prepared in Example 2, with a concentration of 100 mg / ml.

[0137] Experimental methods:

[0138] 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.

[0139] 2. Add 25 μl of test sample solution of different concentrations into the spot tester

[0140] 3. Take photos and record sensory sensations such as itching and tingling according to the test time.

[0141] Results Figure 6 In the figure, a is before treatment, b is after 1 hour of treatment, c is after 2 hours of treatment, and d is after 4 hours of treatment. The upper left, upper right, lower left, and lower right are CK group, unwrapped PDRN-100 group, wrapped PDRN-50 group, and wrapped PDRN-100 group, respectively. The degree of redness, swelling, and itching is shown in Table 10:

[0142] Table 10 Wheal test redness and itching degree

[0143]

[0144] Redness, swelling and itching + indicates the degree, + indicates mild symptoms, ++ indicates moderate symptoms, +++ indicates severe symptoms, and - indicates no symptoms; Tolerability - indicates that it is difficult to tolerate, - indicates that it is tolerable, - indicates only a slight abnormal feeling, and + indicates no adverse feelings.

[0145] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a novel PDRN, 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 tissue material is crushed by an ultrasonic cell disruptor, and then papain is added for enzymatic hydrolysis, and then SDS, a lipid remover and NaCl are added to obtain a tissue mixture, the tissue mixture is stirred, and then centrifuged, and the supernatant is collected, and then the supernatant is mixed with anhydrous ethanol, and allowed to stand, and finally the flocculent DNA chromatographed from the upper layer is taken out, washed, and dissolved in PBS to obtain a crude DNA extract; (2) Cutting and shearing DNA The crude DNA extract is placed in a centrifuge tube and sheared using an ultrasonic shearing device to obtain DNA fragments; (3) Preparation of PDRN by enzymatic hydrolysis Restriction endonucleases and deoxyribonucleases are added to the broken DNA fragments for enzymatic treatment, followed by centrifugation, and the supernatant is collected. The supernatant is mixed with anhydrous ethanol and allowed to stand. Finally, the flocculent DNA from the upper layer is taken out and washed to obtain PDRN.

2. The preparation method of the novel PDRN as claimed in claim 1, characterized in that, 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.

3. The preparation method of the novel PDRN as claimed in claim 1, characterized in that, In step (1), the lipid remover is composed of Tween 20, polyethylene glycol octyl phenyl ether and TE buffer, the amount of Tween 20 is 0.1wt%, the amount of polyethylene glycol octyl phenyl ether is 0.2ml / L, and the amount of TE buffer is 0.5ml / L.

4. The preparation method of the novel 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 1.3-1.6 mol / L, and the specific process of the stirring treatment is stirring in a constant temperature water bath at 37°C for 1 hour.

5. The preparation method of the novel PDRN as claimed in claim 1, characterized in that, 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 500U / ml, and the specific process of the enzymatic treatment is enzymatic hydrolysis in a constant temperature water bath at 37°C for 1 to 2 hours.

6. A new type of PDRN, characterized in that: Prepared according to the preparation method as described in any one of claims 1 to 5.

7. 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 novel PDRN as claimed in claim 6 in a citric acid buffer to obtain a PDRN-citric acid buffer; (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.

8. The preparation method of PDRN supramolecular liposome as claimed in claim 7, characterized in that, The concentration of lipid-ethanol solution was 7.5 mg / ml, the concentration of PDRN-citrate buffer was 0.1 μg / μL, and the synthesis conditions of lipid nanoparticle synthesizer were: inner and outer phase flow rate ratio 1:5, total flow rate 24 ml / min, total product volume 36 ml, front waste liquid 2 ml, and rear waste liquid 0.1 ml.

9. A PDRN supramolecular liposome, characterized in that: Prepared according to the preparation method as claimed in claim 7 or 8.

10. Use of the PDRN as claimed in claim 6 or the PDRN supramolecular liposome as claimed in claim 9 in the preparation of anti-inflammatory drugs or anti-allergic and antipruritic drugs.

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