PDRN-fish collagen composite biological material as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to a PDRN-fish collagen composite biomaterial as well as a preparation method and application thereof. The material is composed of poly (deoxyribonucleotide) (PDRN), low-immunogenicity fish collagen and photo-thermal response type lipidosome, and is a skin repair material with innovativeness and high efficiency. The composite biological material is mainly applied to barrier repair after laser, radio frequency and other photoelectric operations and regeneration treatment of radiodermatitis wounds. Through unique formula design, universal treatment of different injury types can be realized by virtue of triple mechanisms of DNA repair, ECM (extracellular matrix) remodeling and ROS (reactive oxygen species) removal, so that the treatment effect is remarkably improved, and the application value and the unique position in the field of skin repair are important.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, in particular to a PDRN-fish collagen composite biomaterial and a preparation method and application thereof. BACKGROUND

[0002] Skin repair materials are a kind of biomaterials used for promoting wound healing, replacing damaged skin tissue or protecting wounds. The current clinical application of skin repair materials has formed four categories of natural polymers, synthetic polymers, inorganic materials and composite materials. However, due to factors such as material performance, preparation process and biocompatibility, there are still many technical defects in existing products that need to be solved, and it is difficult to fully meet the repair needs of complex wounds.

[0003] Pure collagen dressing: the main component of this type of dressing is bovine or porcine collagen. This type of collagen has a relatively wide source, but has a certain immunogenic risk. Its main role is to provide a physical barrier by forming a physical cover on the wound surface, protecting the wound, reducing direct contact between the wound and the outside world, reducing the risk of infection, and reducing water evaporation to maintain a moist environment for wound healing. It provides a basic condition for wound healing and is suitable for superficial skin damage such as minor abrasions and burns. However, this type of collagen has a relatively wide source, and bovine or porcine collagen has species differences with human collagen, containing a large number of xenoantigen epitopes, which can easily trigger an immune response in the body. The clinical incidence of allergy is as high as 5-12%, and in severe cases, it can cause wound inflammation to worsen, posing a certain immunogenic risk. Moreover, it lacks active ingredients to promote cell proliferation, and cannot fundamentally stimulate cell growth and division, nor can it effectively accelerate the healing process of the wound. For deeper or more complex wounds, the repair effect is not good.

[0004] PDRN gel: As the main active ingredient, PDRN provides active nucleotides to cells through the salvage pathway, promoting cell proliferation and DNA repair; at the same time, it activates the adenosine A2A receptor to inhibit inflammatory response. However, it lacks ECM support and cannot precisely regulate collagen deposition when not combined with a biomimetic collagen matrix. When used alone, the repaired tissue is prone to structural disorder, and the inhibitory effect on inflammation is limited. It has insufficient ability to repair DNA breaks caused by radiation damage and prevent fibrosis. It is mainly used for the adjuvant treatment of wound damage, which can alleviate the damage to skin cells to some extent.

[0005] In addition, existing skin repair materials also have some problems in terms of transdermal efficiency:

[0006] Macromolecular collagen: due to the large molecular weight of collagen, its molecular structure is difficult to penetrate the stratum corneum, a natural barrier of the skin. When used immediately after photoelectric surgery, the amount that can enter the skin and play a role is very small, the bioavailability is low, which seriously limits the exertion of its therapeutic effect, and the repair potential of the material cannot be fully utilized.

[0007] Traditional liposome: the vesicle structure is composed of phospholipid bilayer, and the drug is wrapped inside. The type and proportion of phospholipid will affect the properties of liposome. Although traditional liposome can wrap drugs to improve their stability and transdermal properties, its stability is poor. When the skin temperature is high (such as 45-50℃ after laser surgery), the phospholipid bilayer structure of traditional liposome is easily destroyed, leading to the disintegration of liposome and inactivation, which cannot guarantee the release of drugs at the effective time and site, affecting the treatment effect. Moreover, traditional liposome has insufficient targeting ability and cannot recognize specific temperature areas or damaged sites, which cannot accurately release active ingredients in specific temperature areas, resulting in the release of drugs in non-target areas, reducing the targeting and effectiveness of treatment. SUMMARY

[0008] The purpose of the present application is to provide a PDRN-fish collagen composite biomaterial and its preparation method and application. By synergistically integrating PDRN and low immunogenic deep-sea fish collagen, the anti-inflammatory function of PDRN activating adenosine A2A receptor, the nucleotide supply function of the salvage pathway and the collagen synthesis regulation ability are utilized, combined with the biomimetic ECM structure and low immunogenicity advantage of fish collagen, the problems of unsynchronized PDRN anti-inflammatory and repair, unbalanced collagen synthesis and low transdermal efficiency in existing materials are solved.

[0009] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0010] The present application provides a PDRN-fish collagen composite biomaterial, which comprises polydeoxyribonucleotides, fish collagen and a photo-thermal responsive liposome.

[0011] The mass ratio of the polydeoxyribonucleotides, fish collagen and photo-thermal responsive liposome is 0.5-1.5:3-7:2.5-5.5.

[0012] Optionally, the molecular weight of the polydeoxyribonucleotides is 50-500 bp; and the purity of the polydeoxyribonucleotides is ≥90%.

[0013] Optionally, the molecular weight of the fish collagen is 50-100 kDa.

[0014] Optionally, the preparation method of the fish collagen comprises: extracting fish collagen from deep-sea fish skin by using an ultrasonic-assisted enzymatic process.

[0015] The temperature of the ultrasonic-assisted enzymatic hydrolysis process is 4-10 DEG C, the power of the ultrasonic is 150-200 W, the time of the ultrasonic is 10-15 minutes / time, the interval is 20-40 minutes, and the ultrasonic is performed for 2-4 times.

[0016] Optionally, the average particle size of the photothermal response liposome is 150-220 nm.

[0017] Optionally, the preparation method of the photothermal response liposome comprises the following steps: dissolving soybean lecithin, cholesterol and polyethylene glycol-distearyl phosphatidyl ethanolamine in a mixed solution of chloroform and methanol to form a phospholipid film; adding polydeoxyribonucleotide, fish collagen and phosphate buffer of superoxide dismutase mimetic peptide into the phospholipid film to form a liposome suspension, and subjecting the liposome suspension to high-pressure homogenization to obtain the photothermal response liposome.

[0018] Optionally, the mass ratio of the soybean lecithin, cholesterol and polyethylene glycol-distearyl phosphatidyl ethanolamine is 6:3:2; the volume ratio of the chloroform and methanol is 3-5:1; the mass concentration of the polydeoxyribonucleotide in the phosphate buffer is 0.2-0.5%, the mass concentration of the fish collagen is 1-5%, and the mass concentration of the superoxide dismutase mimetic peptide is 0.01-0.05%; and the high-pressure homogenization is cyclic treatment for 3-5 times under a pressure of 500-1000 bar.

[0019] The application further provides a preparation method of the PDRN-fish collagen composite biomaterial.

[0020] After the polydeoxyribonucleotide, the fish collagen and the photothermal response liposome are mixed, gradient temperature reaction is performed to obtain the PDRN-fish collagen composite biomaterial.

[0021] Optionally, the process parameters of the gradient temperature reaction are as follows: stirring for 5-15 min under the condition of 25-30 DEG C and 800-1200 rpm, then stirring for 10 min under the condition of 35-50 DEG C and 1000-2000 rpm, and finally stirring for 5-15 min under the condition of 35-50 DEG C and 2500-3500 rpm.

[0022] The application further provides application of the PDRN-fish collagen composite biomaterial in preparation of a photodynamic postoperative barrier repair drug and a radioactive dermatitis wound regeneration drug.

[0023] Compared with the prior art, the application has the following beneficial effects:

[0024] Innovation of synergistic repair mechanism: Breakthrough the limitation of single mechanism of existing materials, build a triple synergistic network of "DNA repair-PDRN, ECM remodeling-fish collagen, ROS scavenging-liposome loaded peptide", which is not simply superimposed, but forms a cascade reaction through intermolecular interaction (such as hydrogen bonding between PDRN and collagen, electrostatic adsorption between liposome and collagen fiber), the repair efficiency is higher than the sum of the three components used alone, and this synergistic effect has not been reported in the prior art.

[0025] Precise design of bionic structure: The diameter of fish collagen microfiber network is highly matched with human ECM, and the interpenetrating structure is formed with liposome through gradient warming process, realizing the "dual-phase release" of active ingredients, solving the contradiction of "burst release" or "insufficient sustained release" of traditional materials, and the structure design is unique.

[0026] Temperature accuracy of intelligent delivery: Through the optimization of phospholipid ratio and surface modification, the phase transition temperature of liposome is strictly controlled at 45-48℃, which is completely consistent with the high temperature area after photodynamic therapy, and the targeting of the existing temperature-sensitive liposome (phase transition temperature fluctuation ±5℃) is improved by 60%, realizing "on-demand release".

[0027] Realize the precise regulation of "anti-inflammatory-proliferation-remodeling" three stages: In the acute phase, PDRN-A2A receptor pathway is used to inhibit excessive inflammation, in the medium term, fish collagen scaffold and PDRN nucleotide supply are used to promote cell proliferation, and in the later period, PDRN is used to regulate collagen synthesis to avoid fibrosis, finally significantly improving the effect of barrier repair and radiation dermatitis wound regeneration after photodynamic therapy, and providing a better repair material for clinic. DETAILED DESCRIPTION

[0028] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0029] It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value within the stated range and any other stated value or intermediate value within the stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not an admission that it is prior art.

[0031] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only. It is intended to include all such modifications and variations in the scope of the present application.

[0032] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” and the like are open-ended terms that are intended to denote the inclusion of elements or steps without excluding other elements or steps.

[0033] The raw materials used in the present application can be obtained commercially or prepared by prior art.

[0034] The present application provides a PDRN-fish collagen composite biomaterial, comprising polydeoxyribonucleotides, fish collagen and photothermal responsive liposomes.

[0035] The mass ratio of the polydeoxyribonucleotides, fish collagen and photothermal responsive liposomes is 0.5-1.5:3-7:2.5-5.5, for example, it can be 0.5:7:5.5, 1:5:4 or 1.5:7:4, etc.

[0036] The present application adopts a biomimetic composite system: simulating the PDRN-collagen fiber network structure of the extracellular matrix of the skin, using deep-sea fish collagen to construct a fiber network highly matched with the human ECM, combining the adenosine A2A receptor activation function of polydeoxyribonucleotides, to realize the multidimensional synergy of physical support, signal transmission and inflammation regulation, and more in line with the physiological needs of the natural repair process of the skin.

[0037] An intelligent delivery system is designed: a photothermal responsive liposome is developed, which can sense the high temperature environment of the skin after photoelectric surgery, and accurately target the release of active ingredients in the high temperature zone, avoiding the premature release or waste of active ingredients, and improving the treatment efficiency.

[0038] In the present application, the polydeoxyribonucleotides are derived from salmon testis; the molecular weight of the polydeoxyribonucleotides is 50bp-500bp, for example, can be 50bp, 100bp, 150bp, 200bp, 250bp, 300bp, 350bp, 400bp, 450bp or 500bp, etc.; the purity of the polydeoxyribonucleotides is ≥90%, preferably 99.6%.

[0039] In the present application, polydeoxyribonucleotides (PDRN): as a key active ingredient, have a dual core function: ① as a deoxyribonucleotide precursor, provide active deoxyribonucleotides, nucleosides and bases to cells through a salvage pathway, promote DNA repair and cell viability maintenance; ② specifically activate adenosine A2A receptors, inhibit the release of inflammatory factors (TNF-α, IL-6), and block the secondary damage of excessive inflammatory response to the wound. In cooperation with fish collagen, it can precisely regulate the synthesis rate and proportion of collagen, avoid fibrosis caused by excessive deposition of type I collagen, and keep the ratio of type III / type I collagen at 0.32±0.04 (close to normal skin 0.35), which is significantly better than PDRN treatment alone (ratio 0.18±0.02). Its intermolecular interaction (hydrogen bond) with fish collagen can prolong the residence time of PDRN in the wound to 72 hours, which is 12 times that of pure PDRN gel (6 hours), ensuring continuous function.

[0040] In the present application, PDRN is purified by two-step alcohol precipitation method. A PDRN fragment with a molecular weight of 250bp is obtained, which is verified by experiments to be the interval with the highest repair activity.

[0041] In the present application, the fish collagen is derived from the skin of deep-sea cod, and is subjected to defatting and decellularization treatment; the molecular weight of the fish collagen is 50-100kDa.

[0042] In the present application, low immunogenic fish collagen: derived from deep-sea cod and other cold-water fish skin, forms a synergistic advantage with PDRN: ① provides a biomimetic delivery scaffold for PDRN, with a 50-100nm fiber network matching the human ECM, PDRN is evenly distributed in the collagen fiber gap, avoiding the imbalance caused by excessive local concentration; ② as a target matrix for PDRN to regulate collagen synthesis, the low antigenicity of fish collagen can reduce inflammatory interference and ensure efficient activation of the A2A receptor pathway (TNF-α reduction rate of 68.5%, 40.2% higher than the bovine collagen group); ③ when wrapped together with PDRN by photothermal liposomes, its alpha-helix structure (retention rate 92%) can enhance the stability of the liposomes, making the material structure retention rate at 45°C reach 85%, providing protection for the controlled release of PDRN in the high temperature zone.

[0043] In the present application, the fish collagen preparation method comprises: extracting fish collagen from deep-sea fish skin by using an ultrasonic-assisted enzymatic hydrolysis process.

[0044] The ultrasonic-assisted enzymatic hydrolysis process has a temperature of 4-10℃ (for example, it can be 4℃, 5℃, 6℃, 7℃, 8℃, 9℃ or 10℃, etc.), an ultrasonic power of 150-200W (for example, it can be 150W, 160W, 170W, 180W, 190W or 200W, etc.), an ultrasonic time of 10-15 minutes / time (for example, it can be 10 minutes / time, 11 minutes / time, 12 minutes / time, 13 minutes / time, 14 minutes / time or 15 minutes / time, etc.), an interval of 20-40 minutes (for example, it can be 20 minutes, 25 minutes, 30 minutes, 35 minutes or 40 minutes, etc.), and a total of 2-4 times (for example, it can be 2 times, 3 times or 4 times).

[0045] In some embodiments of the present application, the deep-sea fish skin is subjected to deproteinization treatment before use, and then subjected to enzymatic hydrolysis, filtration and sterilization after enzymatic hydrolysis, and freeze-drying to obtain fish collagen.

[0046] In some embodiments of the present application, a low-temperature enzymatic hydrolysis method is used to extract low-immunogenic collagen from deep-sea fish skin. The ultrasonic-assisted enzymatic hydrolysis process (power 200W, ultrasonic time 10 minutes / time, interval 30 minutes, total 3 times) is introduced innovatively, which, under the condition of 4-10℃, increases the extraction rate of collagen from 65% of the conventional enzymatic hydrolysis method to more than 85%, and improves the integrity of the protein structure, with an α-helix content retention rate of 92%, which is much higher than the 75% of the traditional method. At the same time, affinity chromatography is used to remove glycoprotein impurities in fish collagen, so that the residual amount of sugar chain related to immunogenicity is less than 0.1%.

[0047] In the present application, the average particle size of the photothermal response liposome is 150-220nm, preferably 185±12nm.

[0048] In the present application, the liposome comprises soy lecithin, cholesterol and polyethylene glycol-distearyl phosphatidyl ethanolamine, and further comprises other auxiliary materials such as phosphate buffer and superoxide dismutase mimetic peptide.

[0049] In the present application, the photothermal responsive liposome: composed of phospholipids (soy lecithin: cholesterol: polyethylene glycol-distearyl phosphatidyl ethanolamine = 6:3:2, m / m) of a specific ratio, by introducing polyethylene glycol derivatives to modify the surface of the liposome, the phase transition temperature is accurately controlled at 45-48℃, and it is completely matched with the high temperature area of the skin after photoelectric operation. Stable at normal body temperature (36-37℃), the encapsulation efficiency is maintained above 80%; when it contacts the high temperature area of the skin after photoelectric operation, the release rate can reach 90% within 30 minutes, while the traditional liposome is completely disintegrated within 10 minutes at the same temperature, and cannot realize controllable release. The liposome not only encapsulates PDRN and fish collagen, but also loads superoxide dismutase mimetic peptide, which can synergistically scavenge ROS, solving the problem of insufficient antioxidant capacity of single component.

[0050] In the present application, the preparation method of the photothermal responsive liposome comprises: dissolving soy lecithin, cholesterol and polyethylene glycol-distearyl phosphatidyl ethanolamine in a mixed solution of chloroform and methanol to prepare a phospholipid film; adding a phosphate buffer solution of PDRN, fish collagen and superoxide dismutase mimetic peptide to the phospholipid film to form a liposome suspension, and subjecting the liposome suspension to high-pressure homogenization to obtain the photothermal responsive liposome.

[0051] In the present application, the mass ratio of soy lecithin, cholesterol and polyethylene glycol-distearyl phosphatidyl ethanolamine is 6:3:2; the volume ratio of chloroform and methanol is 3-5:1 (for example, it can be 3:1, 4:1 or 5:1, etc.); the mass concentration of PDRN in the phosphate buffer solution is 0.2-0.5% (for example, it can be 0.2%, 0.3%, 0.4% or 0.5%), the mass concentration of fish collagen is 1-5% (for example, it can be 1%, 2%, 3%, 4% or 5%, etc.), and the mass concentration of superoxide dismutase mimetic peptide is 0.01-0.05% (for example, it can be 0.01%, 0.02%, 0.03%, 0.04% or 0.05%, etc.); the high-pressure homogenization is cyclic treatment for 3-5 times (for example, it can be 3 times, 4 times or 5 times) under a pressure of 500-1000 bar (for example, it can be 500 bar, 600 bar, 700 bar, 800 bar, 900 bar or 1000 bar, etc.).

[0052] In some embodiments of the present application, the photothermal responsive liposome is prepared by using the thin film hydration-high pressure homogenization combined technology. After thin film hydration, high pressure homogenization treatment (pressure 800 bar, cycle 5 times) is introduced to reduce the coefficient of variation of liposome particle size distribution from 25% of the conventional method to below 12%, ensuring batch consistency. After encapsulating PDRN and fish collagen, the zeta potential is stably maintained at-25 mV±3 mV by monitoring with a dynamic light scattering instrument, effectively avoiding the aggregation phenomenon during storage.

[0053] The application also provides a preparation method of the PDRN-fish collagen composite biomaterial.

[0054] The PDRN, fish collagen and the photothermal responsive liposome are mixed, and then a gradient temperature rising reaction is performed to obtain the PDRN-fish collagen composite biomaterial.

[0055] In the application, the process parameters of the gradient temperature rising reaction are as follows: 25-30 DEG C (for example, 25 DEG C, 26 DEG C, 27 DEG C, 28 DEG C, 29 DEG C or 30 DEG C, etc.), 800-1200 rpm (for example, 800 rpm, 900 rpm, 1000 rpm, 1100 rpm or 1200 rpm, etc.) stirring for 5-15 min (for example, 5 min, 8 min, 10 min, 12 min or 15 min, etc.), then rising to 35-50 DEG C (for example, 35 DEG C, 37 DEG C, 40 DEG C, 42 DEG C, 45 DEG C or 50 DEG C, etc.), 1000-2000 rpm (for example, 1000 rpm, 1200 rpm, 1400 rpm, 1500 rpm, 1600 rpm, 1800 rpm or 2000 rpm, etc.) stirring for 5-15 min (for example, 5 min, 8 min, 10 min, 12 min or 15 min, etc.), finally rising to 35-50 DEG C (for example, 35 DEG C, 37 DEG C, 40 DEG C, 42 DEG C, 45 DEG C or 50 DEG C, etc.), 2500-3500 rpm (for example, 2500 rpm, 2800 rpm, 3000 rpm, 3200 rpm or 3500 rpm, etc.) stirring for 5-15 min (for example, 5 min, 8 min, 10 min, 12 min or 15 min, etc.).

[0056] In some embodiments of the application, the preparation of the composite material: PDRN, fish collagen and liposome are mixed in a mass ratio of 1:5:4, and a gradient temperature rising stirring process (25 DEG C→37 DEG C→45 DEG C, stirring for 10 minutes in each stage) is innovatively adopted to promote the fish collagen fiber network and the liposome to form an interpenetrating structure, and the structure can make the active ingredients form a biphasic release curve of 'fast first and slow later' in the release process through cryo-EM observation, 50% is released in the first 6 hours to meet the demand of acute repair, and the remaining 50% is slowly released in the subsequent 72 hours to promote chronic repair, thus solving the problem of single release of traditional mixed materials.

[0057] The application also provides application of the PDRN-fish collagen composite biomaterial in preparation of a photodynamic postoperative barrier repair drug and a radioactive dermatitis wound regeneration drug.

[0058] The composite biomaterial of the application is mainly applied to barrier repair after laser, radio frequency and other photoelectric operations, and regenerative treatment of radioactive dermatitis wounds. Through the unique formula design, it can realize universal treatment of different damage types by means of the triple mechanism of DNA repair, extracellular matrix (ECM) remodeling and reactive oxygen species (ROS) removal, thereby significantly improving the treatment effect and having important application value and unique position in the field of skin repair.

[0059] The technical solutions provided by the application will be described in detail below in combination with embodiments, but they should not be understood as limiting the protection scope of the application.

[0060] Preparation Example 1

[0061] Fish collagen extraction

[0062] S1. The deep-sea cod skin was soaked in 0.1 mol / L NaOH solution for 2 hours to remove impurities, and stirred every 30 minutes to ensure sufficient contact. Then it was repeatedly washed with deionized water until the pH was neutral, cut into 1 cm x 1 cm pieces with a tissue scissors, and stored at -20℃ for standby.

[0063] S2. The frozen fish skin pieces were taken out, and 0.5 mol / L acetic acid solution containing pepsin (2 mg / mL) was added at a solid-liquid ratio of 1:10. Ultrasonic-assisted enzymolysis was carried out in a constant-temperature shaker at 4℃, with ultrasonic power of 200 W, 10 minutes of ultrasonic, 30 minutes of pause, and 3 cycles. The total enzymolysis time was 24 hours. After the enzymolysis was completed, 1 mol / L NaOH was slowly added to adjust the pH to 7.0 to terminate the reaction, and stirring was carried out to avoid local high pH.

[0064] S3. The enzyme solution was centrifuged at 8000 rpm and 4℃ for 15 minutes, and the supernatant was filtered through a 0.22 μm mixed cellulose ester filter membrane to remove bacteria. Then, the concentrated solution was concentrated to 1 / 5 of the original volume at 4℃ using an ultrafiltration membrane with a molecular weight cut-off of 3 kDa. Finally, the concentrated solution was placed in a freeze dryer, pre-frozen at -50℃ for 2 hours, and vacuum freeze-dried for 24 hours to obtain white fluffy fish collagen powder. The molecular weight of the fish collagen powder was mainly distributed in the range of 50-100 kDa as detected by SDS-PAGE electrophoresis. Immunoblotting detection showed that the binding rate of the fish collagen powder to human IgE was reduced by 96% compared with bovine collagen.

[0065] The fish collagen extraction rate was increased from 65% by the conventional enzymolysis method to more than 85%, and the protein structure integrity was improved, with an α-helix content retention rate of 92%, which was much higher than the 75% of the traditional method, and the residual amount of immunogenicity-related sugar chains was less than 0.1%.

[0066] Preparation Example 2

[0067] PDRN purification

[0068] S1. Dissolve PDRN crude in phosphate buffer (pH 7.2) and magnetically stir for 30 minutes to fully dissolve. Prepare a 10 mg / mL solution and filter through a 0.45 μm polyether sulfone filter membrane to remove insoluble impurities.

[0069] S2. Use the AKTA pure protein purification system to load the solution onto a DEAE-Sepharose Fast Flow chromatography column (2.6 cm x 50 cm) at a flow rate of 1 mL / min. After loading, equilibrate the column with phosphate buffer containing 0.1 mol / L NaCl until the baseline is stable, then perform linear gradient elution with phosphate buffer containing 0.1-0.5 mol / L NaCl. The elution flow rate is 1 mL / min, and each tube is collected for 5 mL. Monitor the absorbance at 280 nm, and combine the main peak fractions to obtain PDRN intermediate with a purity of 90%.

[0070] S3. Concentrate the intermediate solution to 5 mg / mL, load onto a Sephadex G-200 chromatography column (1.6 cm x 100 cm), and elute with phosphate buffer at a flow rate of 0.5 mL / min. Each tube is collected for 3 mL, and the 5-8 kDa fraction is selected according to the molecular weight standard curve. The purity of this fraction is 99.6% as determined by high performance liquid chromatography (HPLC). The repair rate of radioactivity DNA damage is 82% as verified by the comet assay, while the repair rate of conventional unsegmented PDRN at the same concentration is only 58%.

[0071] Preparation Example 3

[0072] Liposome preparation

[0073] S1. Dissolve soybean lecithin, cholesterol, and polyethylene glycol-distearyl phosphatidyl ethanolamine in a chloroform-methanol mixture (3:1, v / v) at a mass ratio of 6:3:2, and magnetically stir for 15 minutes to fully dissolve. Transfer the solution to a round-bottom flask, and evaporate on a rotary evaporator at 30°C under reduced pressure for 30 minutes to form a uniform phospholipid film. Place the flask in a vacuum drying oven, and dry at 30°C under vacuum for 2 hours to completely remove residual organic solvents.

[0074] S2. Add phosphate buffer containing PDRN (0.2%, w / v), fish collagen (1%, w / v), and superoxide dismutase mimetic peptide (0.01%, w / v) to the flask, and magnetically stir in a 37°C water bath for 1 hour to hydrate and form a liposome suspension. Transfer the suspension to a high-pressure homogenizer, and further refine the particle size by processing 5 times at 800 bar pressure.

[0075] S3. The above liposome suspension was extruded through 0.45 μm and 0.22 μm polycarbonate membranes, 10 times for each, to obtain uniform photothermal responsive liposomes.

[0076] The average particle size was 185 ± 12 nm, the polydispersity index (PDI) was 0.12, and the zeta potential was stable at -25 mV ± 3 mV, as detected by a dynamic light scattering instrument. The coefficient of variation of the liposome particle size distribution was reduced from 25% for the conventional method to 12%. The drug release rate reached 92% within 30 minutes under 45°C water bath conditions, and the release rate was only 8% within 24 hours under 25°C conditions, which was significantly better than the controlled release performance of traditional liposomes (release rate of 95% within 10 minutes at 45°C).

[0077] Example 1

[0078] PDRN 2 g, fish collagen 10 g, and photothermal responsive liposomes 8 g prepared in the preparation example were weighed into a stirring tank of a double planetary stirrer. First, the components were preliminarily mixed under the conditions of 25°C and 1000 rpm for 10 minutes. Then, the temperature was increased to 37°C, and the stirring was performed at 2000 rpm for 10 minutes to promote intermolecular interaction. Finally, the temperature was increased to 45°C, and the stirring was performed at 3000 rpm for 10 minutes to promote the formation of an interpenetrating network structure. After the stirring was completed, a PDRN-fish collagen composite biomaterial was obtained.

[0079] The physicochemical properties were detected, and the pH value was 6.8-7.2, and the viscosity was 6500 ± 500 cP at 25°C. Rheological detection showed that the storage modulus (G') was 1200 Pa, the loss modulus (G'') was 300 Pa, and G' / G'' = 4.0, indicating good adhesion and mechanical support.

[0080] Comparative Example 1

[0081] PDRN 2 g, fish collagen 10 g, and photothermal responsive liposomes 8 g weighed according to Example 1 were added at one time into a double planetary stirring tank, and stirring was continuously performed at 2000 rpm ± 200 rpm under the condition of constant temperature at 37°C ± 2°C for 30 minutes. The temperature fluctuation was not more than ± 1°C throughout the process, and a non-gradient temperature rising composite biomaterial was obtained after the stirring was completed.

[0082] After the materials prepared in Example 1 and Comparative Example 1 were stored in a 25°C constant temperature box for 3 months, the retention rate of the active ingredients of the material prepared in Example 1 was still 92%, while the retention rate of Comparative Example 1 without the gradient temperature rising process was only 75%.

[0083] Test Example One

[0084] 1. Photodynamic postoperative barrier repair

[0085] 1.1, Experimental animal selection and model establishment: 60 SPF SD rats (body weight 220 ± 20 g, half male and half female) were selected, and after adaptive feeding for 1 week, the back was depilated with an area of about 4 cm x 4 cm; a damage model was made in the depilation area by using a CO2 laser treatment instrument (wavelength 10.6 μm), energy density 10 J / cm 2 , spot diameter 5 mm, 3 damage points were made on the back of each rat with a spacing of 1 cm.

[0086] 1.2, Grouping and treatment: The rats were randomly divided into 3 groups, 10 rats in each group, and the experimental group was smeared with the composite biomaterial (0.1 g / cm 2 ) at the damage site immediately after the operation, the control group 1 was smeared with the same amount of pure collagen dressing (bovine collagen), the control group 2 was smeared with the same amount of PDRN gel (containing PDRN 0.02 g / cm 2 ), and the blank control group was only smeared with normal saline; it was smeared once a day at the same time every day for 7 consecutive days, and the general state of the rats and the changes of the wounds were observed during the period.

[0087] 1.3, Detection index and method:

[0088] Transdermal efficiency: 24 hours after the operation, the FITC-labeled PDRN or collagen was added to the materials in each group by using fluorescence labeling method, the skin tissue was taken 24 hours after smearing, and the transdermal amount was quantitatively analyzed by fluorescence microscope.

[0089] Wound healing rate: 3 days and 7 days after the operation, the Image-Pro Plus 6.0 software was used to calculate the percentage of the wound healing area to the initial area.

[0090] Histological detection: the rats were sacrificed 7 days after the operation, the skin tissue at the damage site was taken, paraffin-embedded section was made, HE staining was used to observe the epidermal thickness and inflammatory cell infiltration, and Masson staining was used to observe the collagen arrangement.

[0091] 2. Regeneration of radioactive dermatitis wound

[0092] 2.1, Clinical case selection: 45 patients with Ⅱ degree radioactive dermatitis (radiotherapy dose 40-60 Gy) were selected, including 15 cases of head and neck tumors, 20 cases of chest tumors, and 10 cases of abdominal tumors; the inclusion criteria: wound area 2-5 cm 2 , no infection, and signed informed consent form; the exclusion criteria: allergic to collagen or PDRN, and combined with autoimmune diseases.

[0093] 2.2, Grouping and treatment: the patients were randomly divided into 3 groups (n = 15), and the experimental group was smeared with the composite biomaterial 2 times (0.2 g / cm 2), control group 1 was smeared with equal amount of pure collagen dressing, and control group 2 was smeared with equal amount of PDRN gel; continuous treatment for 4 weeks, once a week.

[0094] 2.3, detection index and method:

[0095] Wound healing area: transparent grid paper method was used to measure the wound area every week, and the percentage of healing area was calculated.

[0096] DNA damage repair: the edge tissue of the wound was taken, and the number of γ-H2AX positive cells and the number of 53BP1 focus formation were detected by immunofluorescence method.

[0097] ECM remodeling: the expression of type I and type III collagen was detected by immunohistochemical method, and the ratio of type III collagen / type I collagen was calculated.

[0098] Subjective score: visual analogue scale (VAS) was used to evaluate the degree of itching and pain of patients (0-10 points, the higher the score, the more severe the symptoms).

[0099] Safety evaluation: whether there are allergic reactions such as redness, itching, etc. at the drug application site was observed, and the occurrence of adverse reactions was recorded.

[0100] The application test data of photoelectric postoperative barrier repair and radioactive dermatitis wound regeneration are shown in Table 1.

[0101] Table 1 Application test data of photoelectric postoperative barrier repair and radioactive dermatitis wound regeneration

[0102]

[0103]

[0104] As shown in Table 1, the synergistic effect of photo-thermal responsive liposome and biomimetic collagen network solves the problem of transdermal delivery of macromolecular components, and the transdermal efficiency is greatly improved; in the repair of photoelectricity, the wound healing time is shortened by 40% compared with existing materials; in the treatment of radioactive dermatitis, the healing time of Ⅱ degree wound is shortened from an average of 6 weeks to 4 weeks, and the healing quality is better, the scar formation rate is reduced by 75%, and the repair period is significantly shortened. The activation of A2A receptor of PDRN and the low immunogenicity of fish collagen synergize to reduce the level of inflammatory factors by 22.8% compared with PDRN gel 24 hours after operation, to create a suitable microenvironment for wound repair and realize the synchronous improvement of anti-inflammatory-repair; the low immunogenicity of fish collagen significantly reduces the allergic reaction rate compared with bovine collagen dressing (allergic rate 5-8%); the PEG modification of liposome reduces the phagocytosis of macrophages, prolongs the action time and reduces the local stimulation, and the safety is fully guaranteed.

[0105] Comparative example 2

[0106] The difference from Example 1 is only that it does not contain PDRN.

[0107] Comparative Example 3

[0108] The difference from Example 1 is only that it does not contain fish collagen.

[0109] Comparative Example 4

[0110] The difference from Example 1 is only that it does not contain photo-thermal responsive liposomes.

[0111] Test Example Two

[0112] Postoperative photodynamic barrier repair

[0113] Selection of experimental animals and establishment of models: 60 SPF SD rats (body weight 220±20g, half male and half female) were selected, and after adaptive feeding for 1 week, the back was depilated with an area of about 4cm×4cm; a damage model was made in the depilation area using a CO2 laser treatment instrument (wavelength 10.6μm), energy density 10J / cm 2 , spot diameter 5mm, 3 damage points were made on the back of each rat with a spacing of 1cm.

[0114] Grouping and treatment: the rats were randomly divided into 3 groups, 10 rats in each group, and the experimental group was smeared with the composite biomaterial of Example 1 (0.1g / cm 2 ) at the damage site immediately after the operation, the control group 1 was smeared with an equal amount of the composite biomaterial of Comparative Example 2, the control group 2 was smeared with an equal amount of the composite biomaterial of Comparative Example 3, the control group 3 was smeared with an equal amount of the composite biomaterial of Comparative Example 4, and the blank control group was only smeared with normal saline; smearing was performed once a day at the same time every day for 7 consecutive days, and the general state of the rats and the changes in the wound were observed during the period.

[0115] Detection index and method:

[0116] Wound healing rate: on day 3 and day 7 after the operation, the Image-Pro Plus 6.0 software was used to calculate the percentage of the wound healing area to the initial area.

[0117] Histological detection: the rats were sacrificed on day 7 after the operation, the skin tissue at the damage site was taken, paraffin-embedded sectioning was performed, HE staining was used to observe the epidermal thickness and inflammatory cell infiltration, and Masson staining was used to observe the collagen arrangement.

[0118] ECM remodeling: the expression of type I and type III collagens was detected by immunohistochemical method, and the ratio of type III collagen / type I collagen was calculated.

[0119] Table 2 Comparison of 7d data of postoperative photodynamic model

[0120]

[0121] From Table 2, after any single component is missing, the wound healing rate decreases by more than 20%, the ROS clearance ability decreases by about 50%, and the collagen ratio significantly deviates from the normal value.

[0122] When the three components exist simultaneously, each index is significantly better than any double-component combination (p<0.01), confirming that PDRN, fish collagen and photo-thermal responsive liposomes have a synergistic effect.

[0123] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A PDRN-fish collagen composite biomaterial, characterized in that, Including polydeoxyribonucleotides, fish collagen, and photothermal responsive liposomes; The mass ratio of the polydeoxyribonucleotides, fish collagen, and photothermal responsive liposomes is 0.5–1.5:3–7:2.5–5.

5.

2. The PDRN-fish collagen composite biomaterial according to claim 1, characterized in that, The polydeoxyribonucleotide has a molecular weight of 50bp to 500bp; the purity of the polydeoxyribonucleotide is ≥90%.

3. The PDRN-fish collagen composite biomaterial according to claim 1, characterized in that, The molecular weight of the fish collagen is 50-100 kDa.

4. The PDRN-fish collagen composite biomaterial according to claim 1, characterized in that, The method for preparing fish collagen includes: extracting fish collagen from the skin of deep-sea fish using an ultrasound-assisted enzymatic hydrolysis process; The temperature of the ultrasound-assisted enzymatic hydrolysis process is 4-10℃, the power of the ultrasound is 150-200W, the ultrasound time is 10-15 minutes / time, with an interval of 20-40 minutes, for a total of 2-4 times.

5. The PDRN-fish collagen composite biomaterial according to claim 1, characterized in that, The average particle size of the photothermal responsive liposomes is 150–220 nm.

6. The PDRN-fish collagen composite biomaterial according to claim 1, characterized in that, The method for preparing the photothermal responsive liposomes includes: dissolving soybean lecithin, cholesterol, and polyethylene glycol-distearate phosphatidylethanolamine in a mixture of chloroform and methanol to form a phospholipid film; adding polydeoxyribonucleotides, fish collagen, and a phosphate buffer solution of superoxide dismutase mimic peptide to the phospholipid film to form a liposome suspension; and homogenizing the liposome suspension under high pressure to obtain photothermal responsive liposomes.

7. The PDRN-fish collagen composite biomaterial according to claim 6, characterized in that, The mass ratio of soybean lecithin, cholesterol, and polyethylene glycol-distearate phosphatidylethanolamine is 6:3:2; the volume ratio of chloroform and methanol is 3-5:1; the mass concentration of polydeoxyribonucleotides in the phosphate buffer is 0.2-0.5%, the mass concentration of fish collagen is 1-5%, and the mass concentration of superoxide dismutase mimic peptide is 0.01-0.05%; high-pressure homogenization is performed by cycling at 500-1000 bar for 3-5 times.

8. The method for preparing the PDRN-fish collagen composite biomaterial according to any one of claims 1 to 7, characterized in that, The preparation steps include the following: Polydeoxyribonucleotides, fish collagen, and photothermal responsive liposomes were mixed and subjected to a gradient temperature increase reaction to obtain PDRN-fish collagen composite biomaterials.

9. The preparation method according to claim 8, characterized in that, The process parameters for the gradient heating reaction are as follows: stirring at 25-30℃ and 800-1200rpm for 5-15 minutes, then heating to 35-50℃ and stirring at 1000-2000rpm for 5-15 minutes, and finally heating to 35-50℃ and stirring at 2500-3500rpm for 5-15 minutes.

10. The use of the PDRN-fish collagen composite biomaterial according to any one of claims 1 to 7 in the preparation of post-phototherapy barrier repair drugs and radiation dermatitis wound regeneration drugs.

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