A wound dressing and its preparation method and application

Abstract

The present invention belongs to the field of surface skin repair and wound treatment, and specifically relates to a new type of wound dressing, a preparation method and application thereof. The preparation method comprises: preparation and preliminary purification of hemoglobin, fine purification of hemoglobin, degassing treatment, virus inactivation, preparation of wound dressing stock solution and preparation of wound dressing. The wound dressing can be used for wound treatment on the skin surface, especially for the treatment of chronic wounds, especially for the treatment of chronic wounds with delayed healing for more than 8 weeks, especially for ulcers caused by basic metabolic diseases such as diabetes, neuropathic or ischemic ulcers, leg venous insufficiency, ulcers caused by external pressure after skin and tissue aging (such as pressure sores, bedsores), second-degree and below burns, frostbite, etc., especially foot ulcers caused by diabetes; it can also be used for scar repair on the skin surface, and can also be used for repairing difficult-to-heal wounds after surgery.

Description

Technical Field

[0001] The invention belongs to the field of surface skin repair and wound treatment, and specifically relates to a novel wound dressing and a preparation method and application thereof. Background Art

[0002] Chronic wounds are wounds that, due to various factors, cannot achieve anatomical and functional integrity through normal, orderly, and timely repair processes. According to the definition of the German Association for Chronic Wounds (Initiative Chronische Wunden, ICW), wounds that are delayed or stagnant for more than 8 weeks are generally considered chronic wounds. In addition, wounds with underlying diseases that require treatment are also defined as chronic wounds. Common causes include diabetes, peripheral arterial occlusive disease (PAOD), or chronic venous insufficiency (CVI). Although the etiology of wounds varies greatly, the pathophysiological process of chronic wounds almost always includes chronic hypoxia. For example, in patients with PAOD, hypoxia is often caused by arteriosclerotic occlusion of the lower extremity and pelvic arteries. Insufficient blood supply to the affected limb leads to insufficient oxygen supply, resulting in necrosis and ulceration. Once severe limb ischemia occurs, these wounds will have no hope of healing. In contrast, the main feature of CVI is chronic high pressure in the returning veins; the resulting changes in vascular structure can lead to loss of capillaries and reduced density. This can lead to microcirculatory diseases, nutritional changes in the skin, or even leg venous ulcers. In diabetic foot ulcers (DFUs), polyneuropathy with macroangiopathy and functional microangiopathy are often the cause of wound formation.

[0003] The main reason is that the physiological healing process of wounds requires the participation of molecular oxygen, and the demand for energy metabolism and oxygen of wound tissues increases significantly. The human body absorbs oxygen from the outside world (the oxygen concentration in the air is 21%) mainly through the lungs, and the skin absorbs very little. Hemoglobin in red blood cells can transport oxygen to tissue cells throughout the body through the circulatory system. After molecular oxygen is released in the capillaries, it reaches the cells by natural diffusion. During tissue hypoxia, the capillary density decreases, the O2 diffusion distance increases, and the oxygen supply cannot meet the needs of the tissues.

[0004] With the aging of the population and the increasing incidence of diabetes, various chronic wounds such as diabetic foot ulcers, pressure ulcers, lower extremity arteriovenous ulcers, and postoperative wound non-healing are increasing year by year. Among them, the incidence of diabetic foot is high, accounting for almost 1 / 3 of chronic wounds. About 1% to 2% of the world's population will suffer from chronic leg wounds in their lifetime, with an incidence of about 6.3%. And nearly 10 million new chronic wound patients will be added each year. The New England Journal of Medicine published an expert review pointing out that 19% to 34% of diabetic patients will develop diabetic foot ulcers. According to the International Diabetic Foot Organization: The number of amputations caused by diabetic foot exceeds 1 million each year in the world, and one person is amputated due to diabetic foot every 20 seconds.

[0005] The treatment of chronic wounds represented by diabetic foot involves multiple disciplines and requires systematic comprehensive treatment, including blood sugar control, surgical debridement, vascular recanalization, decompression therapy, supportive therapy, etc. Schultz et al. in Germany, based on the healing process of chronic wounds, used wound bed preparation (WBP) as a systematic wound management program and formed a guiding theory for the treatment process of chronic wounds. WBP follows the TIME principle, that is, removing necrotic tissue, controlling infection and inflammation, maintaining a certain moist environment of the wound, and promoting the growth of the wound edge. Among them, controlling wound infection and promoting tissue repair are the key to alleviating diabetic foot and chronic wound symptoms. Treatment techniques include hyperbaric oxygen therapy (HBO) and negative pressure closed drainage technology (NPWT). Hyperbaric oxygen therapy (HBO) exposes the whole body of chronic wound patients to an environment of more than 1 absolute atmosphere (ATA). Pure oxygen enters the lungs through the human respiratory system for gas exchange, and then enters the blood and is transferred to various tissues of the body through circulation. It can enhance the oxygen saturation in the blood, thereby supplying oxygen to chronic wounds, improving the hypoxic state of wound tissue, and accelerating wound healing. Negative pressure closed drainage technology (NPWT) improves wound healing by reducing wound edema, removing bacterial products, and promoting the growth of granulation tissue. However, both hyperbaric oxygen therapy and negative pressure drainage technology require the use of large equipment and professional technicians to accompany and guide, and the treatment time is long, and bedside treatment cannot be achieved. As an auxiliary treatment method, hemoglobin spray can be used by itself at the bedside after being guided by a physician, or it can be used at home under the remote guidance of professional nursing staff. It is quick and convenient and has been widely used. For example, the wound spray patent (CN108752468A), whose main component is porcine hemoglobin, is based on the function of hemoglobin to bind and release oxygen, and the gradient difference of oxygen partial pressure with wound tissue is used to achieve oxygen release. When hemoglobin is sprayed onto the wound, due to the low solubility of oxygen in water, hemoglobin plays the function of transporting oxygen due to its good solubility in water. It can transport O2 from the surrounding air through the wound exudate and enter the bottom of the wound bed, thereby realizing the transport of oxygen molecules and supplying oxygen to the wound bed. The supplied oxygen assists wound healing. In clinical practice, solid dressings, such as breathable or semi-permeable dressings, are generally used to wrap the wound area after using this wound spray.

[0006] However, the preparation route disclosed in the patent (CN108752468A) includes a combination of heating and filtration. It is well known that hemoglobin is easily oxidized into methemoglobin and loses the ability to bind oxygen. Temperature has a great influence on it. Increasing the temperature will accelerate the oxidation rate of hemoglobin, while lowering the temperature can inhibit the oxidation rate of hemoglobin.

[0007] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the invention

[0008] In order to solve the problems existing in the prior art, the present invention provides a method for preparing a novel wound dressing, the preparation method comprising the following steps:

[0009] (1) Preparation and preliminary purification of hemoglobin: Bovine blood is filtered through sodium citrate solution, then lysed with ultrapure water, and then filtered to obtain crude pure hemoglobin;

[0010] (2) Fine purification of hemoglobin: the crude pure hemoglobin is subjected to anion exchange chromatography treatment, and the collected chromatography target eluate is further filtered to obtain finely pure hemoglobin;

[0011] (3) Degassing: the purified hemoglobin is mixed with CO in reverse order to be degassed, and then sterilized and filtered to obtain degassed hemoglobin;

[0012] (4) Virus inactivation: subjecting the degassed hemoglobin to a virus inactivation process to obtain inactivated hemoglobin;

[0013] (5) Preparation of wound dressing stock solution: ultrafiltration of the auxiliary material and the inactivated hemoglobin to obtain a dressing stock solution;

[0014] (6) Preparation of wound dressing: The dressing stock solution is filled to obtain the novel wound dressing.

[0015] In more detail, the preparation method comprises the following steps:

[0016] (I) Preparation and preliminary purification of hemoglobin:

[0017] Filter the bovine blood through a capsule filter, and control the pressure at the liquid inlet end to ≤4psi. Collect the filtered liquid and wash it with a sodium citrate solution, keeping the transmembrane pressure ≤9psi during washing. The collected liquid after washing is lysed with ultrapure water, and the lysed liquid passes through a 100kD hollow fiber membrane filtration system through a peristaltic pump or other power device, and the liquid at the permeate end of the membrane filtration system is collected and passed through a 10kD or 30kD hollow fiber column or membrane package filtration system through a peristaltic pump or other power device, and the liquid at the reflux end is collected. When the conductivity of the collected liquid at the permeate end is ≤1ms / cm, the filtration system is stopped. The liquid collected at the reflux end is passed through a 0.45μm and / or 0.22μm sterile filter to obtain crude pure hemoglobin.

[0018] (II) Fine purification of hemoglobin:

[0019] The crude pure hemoglobin is treated with anion exchange chromatography, the eluents before the elution peak of 15% and after the 10% are discarded, the middle peak is collected, and the hemoglobin concentration is detected. The collected chromatographic target eluent is passed through a 30kD membrane package or a hollow fiber column, and the liquid is replaced with a constant volume of phosphate buffer, and the liquid replacement multiple is maintained at 7-10 times, and pure hemoglobin is obtained after completion.

[0020] (III) Degassing treatment:

[0021] Collect pure hemoglobin, and pump it into the liquid inlet of the degassing device through a medical silicone tube by a peristaltic pump or other power device, and connect the liquid outlet to the silicone tube. Connect the CO cylinder to the air inlet on the degassing device that is opposite to the inlet of the liquid inlet, and connect the air outlet of the degassing device to the air inlet through a three-way pipe. The purpose of this connection is to improve the utilization rate of CO so that the exhausted CO gas can be recycled. Set the peristaltic pump speed and the pressure and flow rate of the CO cylinder outlet within an optimal range. The pure hemoglobin degassed by the degassing membrane is sterilized and filtered at 0.45+0.22μm (i.e., sterilized and filtered through 0.45μm and 0.22μm membranes in sequence, the same below) to obtain degassed hemoglobin.

[0022] (IV) Virus inactivation:

[0023] The virus inactivation method is a low pH inactivation method or a chemical reagent inactivation method. The low pH inactivation method comprises the following steps: adjusting the pH of degassed hemoglobin to 4.0±0.1 with a 100 mM hydrochloric acid solution, storing the degassed hemoglobin in a refrigerator at 2-8°C for 1 hour to 3 days, and detecting the protein content and P50 (the oxygen partial pressure when 50% of the hemoglobin is combined with oxygen. The oxygen partial pressure and P50 do not change, indicating that the low pH condition has no effect on the protein activity); the chemical reagent inactivation method comprises the following steps: adding 0.3% tributyl phosphate (the weight of tributyl phosphate in a certain volume of protein solution) and 0.1%-0.5% sodium cholate (the weight of sodium cholate in a certain volume of protein solution) to the degassed hemoglobin in a certain proportion, placing the degassed hemoglobin in a constant temperature incubator at 22-25°C for 1-6 hours, detecting the protein content and P50, and obtaining the inactivated hemoglobin.

[0024] (V) Preparation of wound dressing stock solution:

[0025] Prepare auxiliary materials and make the pH of auxiliary materials between 5.5 and 6.5;

[0026] The prepared auxiliary material solution is ultrafiltered through a 10kD or 30kD membrane package to replace the inactivated hemoglobin. The replacement multiple is maintained at 6-10 times, and the hemoglobin solution after the replacement is concentrated to the target concentration. The transmembrane pressure of the replacement and concentration steps is controlled at ≤1.5bar, and the peristaltic pump speed during the replacement is controlled at ≤300r / min. The concentrated target protein solution is filtered through a sterile syringe filter into a pre-sterilized vial to obtain the wound dressing stock solution, and the protein content and P50 of the wound auxiliary material stock solution are tested. The tested stock solution is stored at 2-8°C.

[0027] (VI) Preparation of wound dressing:

[0028] Pour the wound dressing stock solution into the filling cup, and control the volume not to exceed 20mL. Physically connect the bottom of the filling cup with the valve of the inflated and sealed dual-package metal bottle (the inflating and sealing pressure is controlled at 4~6bar, and the inflating type is inert gas, air, carbon dioxide, NO, CO, etc.), connect the filling cup with the downward piston of the filling machine, and the power source for driving the downward piston is nitrogen, argon, helium or other inert gas. Adjust the driving pressure to 0.6~0.75MPa, press the control piston rocker, and fill the stock solution in the cup into the dual-package metal bottle through the piston to obtain the wound dressing.

[0029] It should be noted that hemoglobin can also be modified, such as by using a bifunctional reagent such as glutaraldehyde to react chemically with the amino group on the hemoglobin. The ratio of glutaraldehyde to hemoglobin is (28.5±2) mg / g.

[0030] Preferably, in step (1), the sodium citrate solution is filtered and washed 8 to 16 times.

[0031] Preferably, in step (3), the flow rate of pure hemoglobin is 20-1000 mL / min; the CO introduction rate is 1-60 mL / min, and the CO introduction time is 1-210 min.

[0032] Preferably, in step (5), the auxiliary materials include an antibacterial agent, an antioxidant, an electrolyte and a pH adjuster.

[0033] Preferably, the antibacterial agent includes one or a combination of two or more of benzyl alcohol, 1,2-hexanediol, and 2-phenoxyethanol;

[0034] And / or, the antioxidant includes one or a combination of two or more of N-acetylcysteine, proanthocyanidins, cysteine, vitamin C, reduced glutathione, sodium sulfite, sodium dithionite, β-mercaptoethanol, dithiothreitol, and dithioerythritol.

[0035] Preferably, the electrolyte includes one or a combination of two or more of chloride ions, sodium ions, potassium ions, magnesium ions, and calcium ions;

[0036] And / or, the pH adjuster includes one or a combination of two or more of sodium hydroxide, potassium hydroxide, disodium hydrogen phosphate, dipotassium hydrogen phosphate, and sodium dihydrogen phosphate.

[0037] Based on the same technical concept, another embodiment of the present invention is to provide a novel wound dressing obtained by the above preparation method.

[0038] Based on the same technical concept, another solution of the present invention is to provide an application of the novel wound dressing in the preparation of medical cosmetic products.

[0039] It should be emphasized that the wound dressing concentrate can be filled to form a spray, injection, or microcapsule, or can be encapsulated to form microneedles. The wound dressing can be used to treat wounds on the skin surface, especially chronic wounds, especially chronic wounds with delayed healing of more than 8 weeks, especially ulcers caused by underlying metabolic diseases such as diabetes, neuropathic or ischemic ulcers, ulcers caused by leg venous insufficiency, ulcers caused by external pressure after aging of the skin and tissue (such as pressure sores, bedsores), second-degree and below burns, frostbite, etc., especially foot ulcers caused by diabetes; it can also be used for scar repair on the skin surface (such as used as a medical beauty product), and can also be used for the repair of difficult-to-heal wounds after surgery.

[0040] The beneficial effects of the present invention are:

[0041] 1. Taking advantage of the fact that the CO binding efficiency to hemoglobin is about 300 times that of oxygen to hemoglobin, the oxygen dissolved in pure hemoglobin can be easily removed. At the same time, the oxygen removed immediately is circulated back into the liquid channel. Since its ability to bind to hemoglobin is much weaker than that of CO, and the dissolved oxygen content is relatively low in highly purified CO, CO can replace the small amount of oxygen that has been bound to hemoglobin. Secondly, compared with directly passing CO gas into the hemoglobin solution, this can greatly improve the efficiency of CO binding to hemoglobin, because CO gas is insoluble in water, and CO must first overcome the interfacial tension between gas and liquid when it is passed into the protein solution. Using the degassing membrane inside the degassing device for gas exchange can make CO and the protein solution fully contact in reverse, thereby improving the gas-liquid binding efficiency. Another obvious benefit is that the gas passed in is recycled, saving production costs.

[0042] 2. The ability of hemoglobin to bind oxygen and release oxygen is related to P50 (referring to the oxygen partial pressure when 50% of hemoglobin binds to oxygen). The lower the P50, the stronger the ability to bind oxygen, and it is not easy to release oxygen quickly. The higher the P50, the weaker the ability to bind oxygen, and it is not easy to bind oxygen quickly. The present invention controls P50 at a medium level through experiments and formula composition ratio design, balancing the ability to bind and release oxygen (which can be confirmed by the verification data of the present invention).

[0043] 3. Due to the particularity of hemoglobin itself, it is easy to breed bacteria in a high temperature and humidity environment after being sprayed on chronic wounds. Chronic wounds themselves are also prone to wound infection due to the presence of wound exudate (definition of wound infection: According to the definition of the International Wound Infection Institute, wound infection refers to the invasion of wounds by microbial diffusion, causing local, diffuse and / or systemic reactions in the host. Microorganisms multiply in the wound and develop a series of independent factors to overcome the host's defense, resulting in local tissue damage and hindering wound healing. Host defense usually destroys microorganisms unless the host's immune system is compromised or circumvented by microorganisms through a series of measures. Excessive and prolonged inflammatory response, collagen synthesis and epithelialization and tissue damage manifest as wound infection. Therefore, intervention may be required to assist host defense in clearing or destroying invading microorganisms). After wound infection, there are different stages of microorganisms in the wound, ranging from contamination to bacterial colonization, local infection to spread and systemic infection. Clinical management guidelines for infection include: identification of potential biofilms (infection fixation to form biofilms), appropriate selection of cleaning schemes, wound debridement, wound dressings, etc.

[0044] The wound dressing prepared by the present invention can not only utilize the oxygen carrying and releasing capacity of hemoglobin to quickly supply oxygen to chronic wounds by combining with oxygen in the air, provide the oxygen required for chronic wound healing, and support wound healing, but also the dressing is a liquid dressing, which is sprayed onto the wound in the form of aerosol, so that the chronic wound maintains a slightly moist environment. At the same time, the auxiliary material has an inhibitory effect on the biofilm formed by bacterial colonization and growth on the surface of the chronic wound, and has the dual effects of supplying oxygen to the wound and inhibiting the growth of wound bacteria; it has an antibacterial effect on Gram-positive bacteria commonly found in chronic wounds.

[0045] 4. The prepared product can be stored for a long time, thanks to the fact that the efficiency of CO binding to hemoglobin is about 300 times that of oxygen binding to hemoglobin, so that the inside of the product is protected by CO, preventing hemoglobin from being oxidized by oxygen into methemoglobin and losing the ability to bind oxygen; even if the dressing solution is placed in the air for 3 hours, the proportion of hemoglobin oxidized is low, and large-scale production can be achieved under conventional conditions (non-inert gas protection environment), saving production costs (as we all know, the cost of using inert gas is high).

[0046] 5. The product developed by the present invention has the function of auxiliary treatment, and the storage condition is simple, and the refrigerator can be refrigerated. It is light and easy to use, and the operation is simple, and it can be used at the bedside or at home. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0048] Figure 1 It is a schematic diagram of the cracking and crude purification device.

[0049] Figure 2 This is the electrophoresis result diagram in Example 1.

[0050] Figure 3 This is the electrophoresis result diagram in Example 2.

[0051] Figure 4 This is the electrophoresis result diagram in Comparative Example 3.

[0052] Figure 5 This is the state diagram of the sample after heating and centrifugation in Verification Example 1.

[0053] Figure 6 It is a curve chart of wound healing rate in Verification Example 8.

[0054] Figure 7 It is a curve chart of wound healing rate in Verification Example 9.

[0055] Figure 7 Description of special symbols:

[0056] 1) Comparison between the product group and the model group: *P<0.05, there is a significant statistical difference; **P<0.01, there is a very significant statistical difference; ***P<0.001, there is an extremely significant statistical difference;

[0057] 2) Comparison between the positive group and the model group: *P<0.05, there is a significant statistical difference; **P<0.01, there is a very significant statistical difference.

[0058] Figure 8 This is the healing effect diagram of the blank model group (0-23 days after modeling, 5 animals); among them:

[0059] “D” means “day”, such as “D1” means “first day”, and the same below.

[0060] Fig. 9 It is a diagram of the healing effect of the experimental group (Example 1) (0 to 23 days after modeling, 8 animals).

[0061] Fig.10 This is a diagram of the healing effect of the positive control group (control example) (0 to 23 days after modeling, 8 animals).

[0062] Fig.11 It is a curve chart of wound healing rate in Verification Example 10.

[0063] Fig.11 Description of special symbols:

[0064] 1) Comparison between the low-dose group and the model group: *P<0.05, with significant statistical difference;

[0065] 2) Comparison between the positive control group and the model group: #P<0.05, with significant statistical difference.

[0066] Fig.12 This is a rendering of the wound healing process in Verification Example 10.

[0067] The reference numerals in the figure are:

[0068] 1-crude pure hemoglobin; 2-penetration; 3-washing; 4-front of peak; 5-elution peak; 6-peak tail; 7-regeneration; M-molecular weight distribution standard. DETAILED DESCRIPTION

[0069] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0070] Example 1: Preparation of wound dressing using pure hemoglobin

[0071] (I) Preparation and initial purification of hemoglobin

[0072] The collected bovine whole blood was filtered through a capsule filter, and the pressure at the liquid inlet was controlled at 4 psi. The collected filtered liquid was diafiltered with sodium citrate solution (pH = 7.4) at a 10-fold diafiltration rate, and the transmembrane pressure was maintained at 9 psi. The collected liquid after diafiltration was lysed with ultrapure water, and the ratio of ultrapure water to hemoglobin was 1:1.

[0073] The lysed liquid is ultrafiltered through a 100kD hollow fiber membrane filtration system by a peristaltic pump, and the permeate end of the filtration system is closed. The pump speed is set to 250mL / min, circulate for 5min, and then the peristaltic pump is turned off. During the circulation process, the TMP is controlled to be 30psi. After the circulation is completed, the permeate end pipe clamp is opened, the peristaltic pump is turned on, the pump speed is set to 300mL / min, and concentration is started. When the volume of the feed liquid in the reflux end container is reduced to 60% of the volume before concentration, the peristaltic pump is turned off and the concentration is stopped. The peristaltic pump and the peristaltic pump are turned on to start constant volume diafiltration. The pump speeds of the peristaltic pump and the peristaltic pump are adjusted to make the volume of the feed liquid in the liquid storage container 1 constant. During the diafiltration process, the feed liquid in the liquid storage container 1 is taken to detect the protein content. When the hemoglobin content is less than 8% of the hemoglobin content before concentration, the peristaltic pump and the peristaltic pump are turned off to end the diafiltration.

[0074] Close the clamp on the permeate end of the 100kD hollow fiber membrane filter, remove the 100kD hollow fiber membrane filter, connect the infusion and outlet pipelines to the liquid storage container 2, close the permeate end pipeline connected to the 30kD membrane package, open the reflux end pipeline clamp to open the reflux end pipeline, start the peristaltic pump, set the pump speed to 500mL / min, circulate for 5min, and turn off the peristaltic pump. Open the permeate end pipeline clamp, start the peristaltic pump, set the pump speed to 800mL / min, and start concentrating. During the concentration process, take the liquid in the waste liquid bag to test the conductivity. When the conductivity is <1ms / cm, turn off the peristaltic pump and stop concentrating. During the entire concentration process, adjust the flow rate and reflux end pressure to control the inlet end pressure = 0.2MPa. Filter the prepared crude pure hemoglobin through a 0.45+0.22μm sterilizing filter into a sterile container for use. The lysis and crude purification devices such as Figure 1 As shown, crude pure hemoglobin is obtained after completion.

[0075] (II) Fine purification of hemoglobin

[0076] The crude pure hemoglobin was treated with anion exchange chromatography, the pH of the eluent was controlled at 8.2, the first 15% and the last 10% of the eluent of the elution peak were discarded, the middle peak was collected, and the hemoglobin purity was 99% and the yield was 92%. The electrophoresis results were as follows: Figure 2 shown.

[0077] The collected chromatographic target eluate is passed through a 30 kD membrane package or a hollow fiber column, and the liquid is replaced with a phosphate buffer at a constant volume, and the replacement frequency is maintained at 8 times. After completion, pure hemoglobin is obtained.

[0078] (III) Degassing treatment

[0079] 100 mL of pure hemoglobin was collected and pumped into the liquid inlet of the degassing device through a medical silicone tube by a peristaltic pump or other power device, and the liquid outlet was connected to the silicone tube. A CO cylinder was connected to the air inlet of the degassing device opposite to the inlet of the liquid inlet, and the air outlet of the degassing device was connected to the air inlet through a three-way pipe. The peristaltic pump speed was set to 50 mL / min, the CO gas flow rate was set to 40 mL / min, and CO was introduced for 30 minutes. The content of hemoglobin bound to CO (expressed as FCOHb%) was detected. The pure hemoglobin degassed by the degassing membrane was sterilized and filtered through 0.45+0.22 μm, and stored for later use to obtain degassed hemoglobin.

[0080] (IV) Virus inactivation

[0081] The virus inactivation method comprises: adjusting the pH of degassed hemoglobin to 4.0 with a 100 mM hydrochloric acid solution, storing the degassed hemoglobin in a 2° C. refrigerator for 3 hours, and detecting the protein content and P50 to obtain inactivated hemoglobin.

[0082] (V) Preparation of wound dressing stock solution

[0083] Weigh 6 g of 1,2-hexanediol, 6 g of 2-phenoxyethanol, 1 g of N-acetylcysteine, 17.2 g of sodium chloride, 0.63 g of potassium chloride, and 0.67 g of calcium chloride into a beaker, dissolve them with an appropriate amount of purified water, transfer them to a 2 L Schott bottle, add purified water to close to the scale, place the Schott bottle on a magnetic stirrer, and adjust the pH of the solution to 6.5 with 2 M sodium hydroxide solution.

[0084] The prepared auxiliary material solution was used to ultrafilter the inactivated hemoglobin through a 10kD membrane package. The replacement multiple was maintained at 10 times. The transmembrane pressure of the membrane package during the replacement process was 1.2 bar. The hemoglobin solution after the replacement was concentrated to 10g / dL. The transmembrane pressure during the concentration process was 1.5 bar, and the peristaltic pump speed was 280r / min. The concentrated target protein solution was filtered through a sterile syringe filter into a pre-sterilized vial to obtain the wound dressing stock solution, and the protein content and P50 of the wound dressing stock solution were tested.

[0085] (VI) Preparation of wound dressing

[0086] Pour the wound dressing stock solution into the filling cup, and control the volume not to exceed 20mL. Physically connect the bottom of the filling cup with the valve of the inflated and sealed dual-package metal bottle (the inflating and sealing pressure is 4.5bar, and the inflating gas is nitrogen), connect the filling cup with the downward piston of the filling machine, and the power source for driving the downward piston is nitrogen. Adjust the driving pressure to 0.75MPa, press the control piston rocker, and fill the stock solution in the cup into the dual-package metal bottle through the piston to obtain the wound dressing.

[0087] Example 2: Preparation of wound dressing by polymerizing hemoglobin

[0088] (I) Preparation and initial purification of hemoglobin

[0089] The collected bovine whole blood was filtered through a capsule filter, and the pressure at the liquid inlet was controlled at 3 psi. The collected filtered liquid was diafiltered with sodium citrate solution (pH 7.4) at a 10-fold diafiltration rate, and the transmembrane pressure was maintained at 8 psi. The collected diafiltration liquid was lysed with ultrapure water, and the ratio of ultrapure water to hemoglobin was 1:1.

[0090] The lysed liquid passes through a 100kD hollow fiber membrane filtration system through a peristaltic pump, and the permeate end of the filtration system is closed. The pump speed is set to 300mL / min, circulates for 5min, and then the peristaltic pump is turned off. During the circulation process, TMP is controlled to be 20psi. After the circulation is completed, the permeate end pipe clamp is opened, the peristaltic pump is turned on, the pump speed is set to 200mL / min, and concentration is started. When the volume of the feed liquid in the reflux end container is reduced to 60% of the volume before concentration, the peristaltic pump is turned off to stop concentration. The peristaltic pump and the peristaltic pump are turned on to start constant volume diafiltration. The pump speeds of the peristaltic pump and the peristaltic pump are adjusted to make the volume of the feed liquid in the liquid storage container 1 constant. During the diafiltration process, the feed liquid in the liquid storage container 1 is taken to test the protein content. When the hemoglobin content is less than 8% of the hemoglobin content before concentration, the peristaltic pump and the peristaltic pump are turned off to end the diafiltration.

[0091] Close the 100kD hollow fiber membrane filter permeate end pipe clamp, remove the 100kD hollow fiber membrane filter, connect the infusion and outlet pipes to the liquid storage container 2, close the permeate end pipe clamp connected to the 30kD membrane package, open the reflux end pipe clamp to open the reflux end pipe, start the peristaltic pump, set the pump speed to 1000mL / min, circulate for 5min, and turn off the peristaltic pump. Open the permeate end pipe clamp, start the peristaltic pump, set the pump speed to 500mL / min, and start concentration. During the concentration process, take the material liquid in the waste liquid bag to test the conductivity. When the conductivity is less than 1ms / cm, turn off the peristaltic pump and stop concentrating. During the entire concentration process, adjust the flow rate and reflux end pressure to control the inlet end pressure = 0.15MPa. Filter the prepared crude pure hemoglobin through a 0.45+0.22μm sterilizing filter into a sterile container for standby use, and obtain crude pure hemoglobin after completion.

[0092] (II) Fine purification of hemoglobin

[0093] The crude pure hemoglobin was treated with anion exchange chromatography, the pH of the eluent was controlled at 8.2, the first 15% and the last 10% of the eluent were discarded, the middle peak was collected, and the hemoglobin purity was 99% and the yield was 91%. The electrophoresis results are as follows Figure 3 shown.

[0094] The collected chromatographic target eluate is passed through a 30 kD membrane package or a hollow fiber column, and the liquid is replaced with a phosphate buffer at a constant volume, and the liquid replacement multiple is maintained at 7 times. After completion, pure hemoglobin is obtained.

[0095] (III) Polymerization

[0096] Add pure hemoglobin to the polymerization reactor, turn on the peristaltic pump, connect the pipe on the peristaltic pump to the ultrapure water storage bag, set the pump speed, and pump ultrapure water into the polymerization reactor. Take samples to test the total hemoglobin concentration. When the total hemoglobin (ctHb) concentration is 2.5g / dL, turn off the peristaltic pump. Turn on the magnetic stirrer and set the speed to 200mL / min.

[0097] Connect the circulating water bath to the polymerization jacket, turn on the circulating water bath, set the temperature to (45±1)℃, and start heating the feed liquid in the polymerization reactor. When the thermometer indicates (40±2)℃, prepare a working solution with a glutaraldehyde concentration of 6mg / mL according to the addition ratio of glutaraldehyde solution (28.5)mg / g pure hemoglobin, control the glutaraldehyde addition time to 40min, divide the volume of the prepared glutaraldehyde working solution by the addition time to obtain the average addition flow rate, set the corresponding flow rate on the peristaltic pump, turn on the peristaltic pump until all the required glutaraldehyde working solution is added to the feed liquid, then turn off the peristaltic pump and keep stirring for 20min. Switch the circulating water in the polymerization reactor jacket, set the circulating water bath temperature to 4℃ cooling water, and stop the circulating water bath when the polymerization reactor thermometer indicates a drop to <29℃. Connect the sodium borate working solution (pH=12) storage bag to the pipeline on the peristaltic pump, turn on the peristaltic pump, set the pump speed to 50mL / min, add the sodium borate working solution to the polymerization reactor, sample and test the pH of the feed solution in the polymerization reactor, and turn off the peristaltic pump when pH=10.2. Sample and test the hemoglobin concentration. Prepare a working solution with a sodium borohydride concentration of 7.0mg / mL according to the ratio of 10mg / g pure hemoglobin, connect the sodium borohydride working solution to the pipeline on the peristaltic pump, control the addition time of the sodium borohydride working solution to 15min, divide the volume of the prepared sodium borohydride working solution by the addition time to obtain the average addition flow rate, set the corresponding flow rate on the peristaltic pump, turn on the peristaltic pump and add the sodium borohydride solution to the polymerization reactor until all the working solution has been added. Concentrate the hemoglobin solution after polymerization to 12g / dL through a 30kd membrane package, and then replace the solution 8 times with a phosphate buffer with a pH=7.6. The hemoglobin solution after the liquid replacement is sterilized and filtered through 0.45+0.22 μm to obtain modified pure hemoglobin.

[0098] (IV) Degassing

[0099] Collect 100 mL of modified pure hemoglobin, pump the modified pure hemoglobin into the liquid inlet of the degassing device through a medical silicone tube by a peristaltic pump or other power device, and connect the liquid outlet to the silicone tube. Connect the CO cylinder to the air inlet on the degassing device that is opposite to the inlet of the liquid inlet, connect the air outlet of the degassing device to the air inlet through a three-way pipe, set the peristaltic pump speed to 50 mL / min, the CO gas flow rate to 40 mL / min, pass CO for 30 minutes, detect the content of hemoglobin bound to CO (expressed as FCOHb%), and the pure hemoglobin degassed by the degassing membrane is sterilized and filtered through 0.45+0.22μm, and stored for later use to obtain degassed hemoglobin.

[0100] (V) Virus inactivation

[0101] The virus inactivation method comprises: adjusting the pH of degassed hemoglobin to 4.0 with a 100 mM hydrochloric acid solution, storing the degassed hemoglobin in a 5° C. refrigerator for 3 hours, and detecting the protein content and P50 to obtain inactivated hemoglobin.

[0102] (VI) Preparation of wound dressing stock solution

[0103] Weigh 12.2 g of 1,2-hexanediol, 1 g of N-acetylcysteine, 17.2 g of sodium chloride, 0.63 g of potassium chloride, and 0.67 g of calcium chloride into a beaker, dissolve them with an appropriate amount of purified water, transfer them to a 2 L Schott bottle, add purified water to close to the scale, place the Schott bottle on a magnetic stirrer, and adjust the pH of the solution to 6.5 with 2 M sodium hydroxide solution.

[0104] The prepared auxiliary material solution was used to ultrafilter the inactivated hemoglobin through a 10kD membrane package. The replacement multiple was maintained at 8 times. The transmembrane pressure of the membrane package during the replacement process was 1.5 bar. The hemoglobin solution after the replacement was concentrated to 10g / dL. The transmembrane pressure during the concentration process was 1.5 bar, and the peristaltic pump speed was 300r / min. The concentrated target protein solution was filtered through a sterile syringe filter into a pre-sterilized vial to obtain the wound dressing stock solution, and the protein content and P50 of the wound dressing stock solution were tested.

[0105] (VII) Preparation of wound dressings

[0106] Pour the wound dressing stock solution into the filling cup, and control the volume not to exceed 20mL. Physically connect the bottom of the filling cup with the valve of the inflated and sealed dual-package metal bottle (the inflating and sealing pressure is 4.5bar, and the inflating gas is nitrogen), connect the filling cup with the downward piston of the filling machine, and the power source for driving the downward piston is nitrogen. Adjust the driving pressure to 0.7MPa, press the control piston rocker, and fill the stock solution in the cup into the dual-package metal bottle through the piston to obtain the wound dressing.

[0107] Comparative Example 1: Preparation of wound dressing using pure hemoglobin without CO binding (compared with Example 1)

[0108] (I) Preparation and initial purification of hemoglobin

[0109] The collected bovine whole blood was filtered through a capsule filter, and the pressure at the liquid inlet was controlled to be 4 psi. The filtered liquid was collected and diafiltered with a sodium citrate solution (pH 7.4) at a 10-fold diafiltration rate, and the transmembrane pressure was maintained at 9 psi. The collected liquid after diafiltration was lysed with ultrapure water, and the ratio of ultrapure water to hemoglobin was 1:1.

[0110] The lysed liquid passes through a 100kD hollow fiber membrane filtration system through a peristaltic pump, and the permeate end of the filtration system is closed. The pump speed is set to 150mL / min, circulate for 5min, and then the peristaltic pump is turned off. During the circulation process, the TMP is controlled to be 25psi. After the circulation is completed, the permeate end pipe clamp is opened, the peristaltic pump is turned on, the pump speed is set to 200mL / min, and concentration is started. When the volume of the feed liquid in the reflux end container is reduced to 60% of the volume before concentration, the peristaltic pump is turned off to stop concentration. The peristaltic pump and the peristaltic pump are turned on to start constant volume diafiltration. The pump speeds of the peristaltic pump and the peristaltic pump are adjusted to make the volume of the feed liquid in the liquid storage container 1 constant. During the diafiltration process, the feed liquid in the liquid storage container 1 is taken to test the protein content. When the hemoglobin content is less than 8% of the hemoglobin content before concentration, the peristaltic pump and the peristaltic pump are turned off to end the diafiltration.

[0111] Close the 100kD hollow fiber membrane filter permeate end pipe clamp, remove the 100kD hollow fiber membrane filter, connect the infusion and outlet pipes to the liquid storage container 2, close the permeate end pipe clamp connected to the 30kD membrane package, open the reflux end pipe clamp to open the reflux end pipe, start the peristaltic pump, set the pump speed to 500mL / min, circulate for 5min, and turn off the peristaltic pump. Open the permeate end pipe clamp, start the peristaltic pump, set the pump speed to 700mL / min, and start concentration. During the concentration process, take the material liquid in the waste liquid bag to test the conductivity. When the conductivity is <1ms / cm, turn off the peristaltic pump and stop concentrating. During the entire concentration process, adjust the flow rate and reflux end pressure to control the inlet end pressure = 0.2MPa. Filter the prepared crude pure hemoglobin through a 0.45+0.22μm sterilizing filter into a sterile container for standby use, and obtain crude pure hemoglobin after completion.

[0112] (II) Fine purification of hemoglobin

[0113] The crude pure hemoglobin was subjected to anion exchange chromatography, the pH of the eluent was controlled at 8.2, the first 15% and the last 10% of the eluent of the elution peak were discarded, the middle peak was collected, and the hemoglobin purity was detected to be 99%, and the yield was 90%.

[0114] The collected chromatographic target eluate is passed through a 30 kD membrane package or a hollow fiber column, and the liquid is replaced with a phosphate buffer at a constant volume, and the liquid replacement multiple is maintained at 9 times. After completion, pure hemoglobin is obtained.

[0115] (III) Degassing treatment

[0116] Collect 100 mL of the liquid at the reflux end of the second step, and pump the reflux end liquid into the liquid inlet of the degassing device through a medical silicone tube by a peristaltic pump or other power device, and connect the liquid outlet to the silicone tube. Connect a nitrogen cylinder to the air inlet of the degassing device opposite to the inlet of the liquid inlet, connect the air outlet of the degassing device to the air inlet through a three-way pipe, set the peristaltic pump speed to 50 mL / min, the nitrogen gas flow rate to 50 mL / min, and introduce nitrogen for 30 minutes, detect the hemoglobin content, and the pure hemoglobin degassed by the degassing membrane is sterilized and filtered by 0.45+0.22 μm, and stored for later use to obtain degassed hemoglobin.

[0117] (IV) Virus inactivation

[0118] The virus inactivation method is low pH, and the steps of the low pH method are to adjust the pH of the hemoglobin prepared in step three to 4.0 with a 100mM hydrochloric acid solution, store it in a refrigerator at 2-8°C for 3h, detect the protein content and P50, and obtain inactivated hemoglobin.

[0119] (V) Preparation of wound dressing stock solution

[0120] Weigh 12.1 g of 2-phenoxyethanol, 1 g of N-acetylcysteine, 17.2 g of sodium chloride, 0.63 g of potassium chloride, and 0.67 g of calcium chloride into a beaker, dissolve them with an appropriate amount of purified water, transfer them to a 2 L Schott bottle, add purified water to close to the scale, place the Schott bottle on a magnetic stirrer, and adjust the pH of the solution to 6.5 with 2 M sodium hydroxide solution.

[0121] The prepared auxiliary material solution was used to ultrafilter the inactivated hemoglobin through a 10kD membrane package. The replacement multiple was maintained at 10 times. The transmembrane pressure of the membrane package during the replacement process was 1.0 bar. The hemoglobin solution after the replacement was concentrated to 10g / dL. The transmembrane pressure during the concentration process was 1.4 bar, and the peristaltic pump speed was 250r / min. The concentrated target protein solution was filtered through a sterile syringe filter into a pre-sterilized vial to obtain the wound dressing stock solution, and the protein content and P50 of the wound dressing stock solution were tested.

[0122] (VI) Preparation of wound dressing

[0123] Pour the wound dressing stock solution into the filling cup, and control the volume not to exceed 20mL. Physically connect the bottom of the filling cup with the valve of the inflated and sealed dual-package metal bottle (the inflating and sealing pressure is 4.5bar, and the inflating gas is nitrogen), connect the filling cup with the downward piston of the filling machine, and the power source for driving the downward piston is nitrogen. Adjust the driving pressure to 0.75MPa, press the control piston rocker, and fill the stock solution in the cup into the dual-package metal bottle through the piston to obtain the wound dressing.

[0124] Comparative Example 2: Preparation of wound dressing using polymerized hemoglobin without CO binding (compared with Example 2)

[0125] (I) Preparation and initial purification of hemoglobin

[0126] The collected bovine whole blood was filtered through a capsule filter, and the pressure at the liquid inlet was controlled at 3 psi. The collected filtered liquid was diafiltered with sodium citrate solution (pH 7.4) at a 10-fold diafiltration rate, and the transmembrane pressure was maintained at 7 psi. The collected liquid after diafiltration was lysed with ultrapure water, and the ratio of ultrapure water to hemoglobin was 1:1.

[0127] The lysed liquid passes through a 100kD hollow fiber membrane filtration system through a peristaltic pump, and the permeate end of the filtration system is closed. The pump speed is set to 150mL / min, circulate for 5min, and then the peristaltic pump is turned off. During the circulation process, TMP is controlled to be 18psi. After the circulation is completed, the permeate end pipe clamp is opened, the peristaltic pump is turned on, the pump speed is set to 190mL / min, and concentration is started. When the volume of the feed liquid in the reflux end container is reduced to 60% of the volume before concentration, the peristaltic pump is turned off to stop concentration. The peristaltic pump and the peristaltic pump are turned on to start constant volume diafiltration. The pump speeds of the peristaltic pump and the peristaltic pump are adjusted to keep the volume of the feed liquid in the liquid storage container 1 constant. During the diafiltration process, the feed liquid in the liquid storage container 1 is taken to detect the protein content. When the hemoglobin content is less than 8% of the hemoglobin content before concentration, the peristaltic pump and the peristaltic pump are turned off to end the diafiltration.

[0128] Close the 100kD hollow fiber membrane filter permeate end pipe clamp, remove the 100kD hollow fiber membrane filter, connect the infusion and outlet pipes to the liquid storage container 2, close the permeate end pipe clamp connected to the 30kD membrane package, open the reflux end pipe clamp to open the reflux end pipe, start the peristaltic pump, set the pump speed to 600ml / min, circulate for 5min, and turn off the peristaltic pump. Open the permeate end pipe clamp, start the peristaltic pump, set the pump speed to 800ml / min, and start concentration. During the concentration process, take the material liquid in the waste liquid bag to test the conductivity. When the conductivity is less than 1ms / cm, turn off the peristaltic pump and stop concentrating. During the entire concentration process, adjust the flow rate and reflux end pressure to control the inlet end pressure = 0.2MPa. Filter the prepared crude pure hemoglobin through a 0.45+0.22μm sterilizing filter into a sterile container for standby use. After completion, obtain crude pure hemoglobin.

[0129] (II) Fine purification of hemoglobin

[0130] The crude pure hemoglobin was treated by anion exchange chromatography, the pH of the eluent was controlled at 8.2, the first 15% and the last 10% of the eluent of the elution peak were discarded, the middle peak was collected, and the hemoglobin purity was detected to be 99%, and the yield was 92%.

[0131] The collected chromatographic target eluate is passed through a 30 kD membrane package or a hollow fiber column, and the liquid is replaced with a phosphate buffer at a constant volume, and the liquid replacement multiple is maintained at 7 times. After completion, pure hemoglobin is obtained.

[0132] (III) Polymerization

[0133] Add pure hemoglobin to the polymerization reactor, turn on the peristaltic pump, connect the pipe on the peristaltic pump to the ultrapure water storage bag, set the pump speed, and pump ultrapure water into the polymerization reactor. Take samples to test the total hemoglobin concentration. When the total hemoglobin (ctHb) concentration is 3.5g / dL, turn off the peristaltic pump. Turn on the magnetic stirrer and set the speed to 200mL / min.

[0134] Connect the circulating water bath to the polymerization jacket, turn on the circulating water bath, set the temperature to 44°C, and start heating the feed liquid in the polymerization reactor. When the thermometer indicates that it reaches 40°C, prepare a working solution with a glutaraldehyde concentration of 6 mg / mL according to the addition ratio of glutaraldehyde solution (30) mg / g pure hemoglobin, control the glutaraldehyde addition time to 45 minutes, and divide the volume of the prepared glutaraldehyde working solution by the addition time to obtain the average addition flow rate. Set the corresponding flow rate on the peristaltic pump, turn on the peristaltic pump until all the required glutaraldehyde working solution is added to the feed liquid, then turn off the peristaltic pump and keep stirring for 20 minutes. Switch the circulating water in the polymerization reactor jacket, set the circulating water bath temperature to 4°C cooling water, and stop the circulating water bath when the polymerization reactor thermometer indicates a drop to <29°C. Connect the sodium borate working solution (pH=12) storage bag to the pipeline on the peristaltic pump, turn on the peristaltic pump, set the pump speed to 100mL / min, add the sodium borate working solution to the polymerization reactor, sample and detect the pH of the feed solution in the polymerization reactor, and turn off the peristaltic pump when pH=10. Sample and detect the hemoglobin concentration. Prepare a working solution with a sodium borohydride concentration of 7.0mg / mL according to the ratio of 10mg / g pure hemoglobin, connect the sodium borohydride working solution to the pipeline on the peristaltic pump, control the addition time of the sodium borohydride working solution to 20min, divide the volume of the sodium borohydride working solution after preparation by the addition time to obtain the average addition flow rate, set the corresponding flow rate on the peristaltic pump, turn on the peristaltic pump to add the sodium borohydride solution to the polymerization reactor until all the working solution has been added. Concentrate the hemoglobin solution after polymerization to 13g / dL through a 30kd membrane package, and then replace the solution 9 times with a phosphate buffer with a pH=7.6. The hemoglobin solution after the liquid replacement is sterilized and filtered through 0.45+0.22 μm to obtain pure hemoglobin.

[0135] (IV) Degassing

[0136] Collect 100 mL of the liquid at the reflux end of the second step, and pump the reflux end liquid into the liquid inlet of the degassing device through a medical silicone tube by a peristaltic pump or other power device, and connect the liquid outlet to the silicone tube. Connect a nitrogen cylinder to the air inlet of the degassing device opposite to the inlet of the liquid inlet, connect the air outlet of the degassing device to the air inlet through a three-way pipe, set the peristaltic pump speed to 50 mL / min, the gas flow rate to 40 mL / min, and pass the gas for 30 minutes, detect the hemoglobin (expressed as FCOHb%) content, and the pure hemoglobin degassed by the degassing membrane is sterilized and filtered through 0.45+0.22μm, and stored for later use to obtain degassed hemoglobin.

[0137] (V) Virus inactivation

[0138] The virus inactivation method comprises: adjusting the pH of degassed hemoglobin to 4.0 with a 100 mM hydrochloric acid solution, storing the degassed hemoglobin in a 4° C. refrigerator for 3 hours, and detecting the protein content and P50 to obtain inactivated hemoglobin.

[0139] (VI) Preparation of wound dressing stock solution

[0140] Weigh 12.1 g of 1,2-hexanediol, 1 g of N-acetylcysteine, 17.2 g of sodium chloride, 0.63 g of potassium chloride, and 0.67 g of calcium chloride into a beaker, dissolve them with an appropriate amount of purified water, transfer them to a 2 L Schott bottle, add purified water to close to the scale, place the Schott bottle on a magnetic stirrer, and adjust the pH of the solution to 6.5 with 2 M sodium hydroxide solution.

[0141] The prepared auxiliary material solution was used to ultrafilter the inactivated hemoglobin through a 10kD membrane package. The replacement multiple was maintained at 10 times. The transmembrane pressure of the membrane package during the replacement process was 1.0 bar. The hemoglobin solution after the replacement was concentrated to 10g / dL. The transmembrane pressure during the concentration process was 1.4 bar, and the peristaltic pump speed was 250r / min. The concentrated target protein solution was filtered through a sterile syringe filter into a pre-sterilized vial to obtain the wound dressing stock solution, and the protein content and P50 of the wound dressing stock solution were tested.

[0142] (VII) Preparation of wound dressings

[0143] Pour the wound dressing stock solution into the filling cup, and control the volume not to exceed 20mL. Physically connect the bottom of the filling cup with the valve of the inflated and sealed dual-package metal bottle (the inflating and sealing pressure is 4.5bar, and the inflating gas is nitrogen), connect the filling cup with the downward piston of the filling machine, and the power source for driving the downward piston is nitrogen. Adjust the driving pressure to 0.7MPa, press the control piston rocker, and fill the stock solution in the cup into the dual-package metal bottle through the piston to obtain the wound dressing.

[0144] Comparative Example 3

[0145] Similar products, hemoglobin is isolated from whole blood of pigs by separating red blood cells from serum, lysing the collected red blood cells, granulating the cell fragments, obtaining hemoglobin carrying CO by introducing CO gas until the liquid sample is saturated, heating the solution, performing several filtration steps, including a virus filtration step and washing the obtained hemoglobin solution by adding 2 volumes of 0.9% saline twice, and filtering the solution. A ready-to-use composition for wound treatment is prepared, which contains 10% purified and stabilized hemoglobin dissolved in 0.9% NaCl, 0.05% N-acetylcysteine ​​and 0.7% phenoxyethanol. The composition is again filled with CO gas, divided into 10 portions, and loaded into spray cans respectively.

[0146] The product of comparative example 3 was subjected to electrophoresis analysis after being sprayed out, and the protein purity was about 95%. Figure 4 shown.

[0147] Verification Example 1: Stability of hemoglobin under different treatment conditions

[0148] 6 mL of the wound dressing stock solution (concentration of 13 g / dL) prepared in Example 1 was equally divided into two portions in 50 mL containers, the first portion was recorded as 1#, and the second portion was recorded as 2#. The 2# sample was placed in 65°C water for 1 hour, and was manually shaken during the placement. Then the two heated samples were placed in a high-speed refrigerated centrifuge at a speed of 10000×g, T=4°C, and t=10min. The heated and centrifuged samples and the untreated stock solution were taken to detect the hemoglobin content, and the results are shown in Table 1. Figure 5 This is the state of the sample after heating centrifugation.

[0149] Table 1

[0150]

[0151] Note:

[0152] ctHb——indicates total hemoglobin content;

[0153] FCOHb——denotes the content of combined CO hemoglobin;

[0154] FMetHb - indicates the content of oxygenated hemoglobin.

[0155] From the protein concentration and sample status, we can see that: Sample 1# was bound to CO, and Sample 2# was heated and centrifuged after being bound to CO. The protein concentration after heating changed significantly, decreasing by about 31%, and the proportion of methemoglobin increased by nearly 11%. This shows that the binding of CO and heating also have a relatively large impact on the stability of hemoglobin.

[0156] Verification Example 2: Comparison of results of hemoglobin inactivation by low pH and chemical reagents

[0157] 20 mL of the hemoglobin solution prepared by the degassing step in Example 1 was adjusted to pH 4.0 with 0.1 M hydrochloric acid solution, and the amount of hydrochloric acid added when adjusting to the target pH was about 20 mL. The sample after pH adjustment was immediately taken to test the protein concentration, and the sample was placed in a refrigerator at 2-8°C for 3 days, and the protein content was tested at 3h, 6h, and 3 days, respectively. The results are shown in Table 2.

[0158] Table 2

[0159]

[0160] From the results, it can be seen that the total concentration of hemoglobin did not change before and after degassing, and the total hemoglobin concentration after adjustment dropped to half of that before adjustment. The reason is that the total volume of hydrochloric acid added at the end of pH adjustment is equal to the volume of hemoglobin before adjustment, and the hemoglobin is diluted by half. This shows that the hemoglobin solution adjusted to pH = 4.0 and left for 3 hours has no effect on the total protein concentration.

[0161] 0.3% tributyl phosphate and 0.2% sodium cholate solution were added to 20 mL of the hemoglobin solution prepared by the degassing step in Example 1. The solution was kept in a stirring state (stirring speed was 100 r / min) before addition. After addition, stirring was continued for 20 minutes. Stirring was stopped and the solution was placed in a 24°C incubator for 12 hours. The protein concentration was detected at 6 hours and 12 hours, respectively. The results are shown in Table 3.

[0162] Table 3

[0163]

[0164] From the results, it can be seen that the total protein concentration of the protein solution after CO2 combination was treated with chemical reagents for 6 hours, and the total protein concentration continued to decrease after being placed for 6 hours, indicating that the added chemical reagents have an effect on the stability of hemoglobin. Based on the above results, the pH method was used to inactivate the degassed hemoglobin.

[0165] The degassed hemoglobin solution prepared in Example 1 was verified by a third party according to YY / T 0771.3-2009 Animal-derived Medical Devices Part 3: Confirmation Method for Removal and Inactivation of Viruses and Transmissible Spongiform Encephalopathy (TSE) Factors. The added indicator viruses included poliovirus Sabin type 1 (PV1, non-enveloped RNA virus), bovine viral diarrhea virus (BVDV, enveloped RNA virus), and pseudorabies virus (PRV, enveloped DNA virus). After adding the indicator viruses, a low pH inactivation step was performed. The experimental results are shown in Table 4.

[0166] Table 4

[0167]

[0168] The results showed that the low pH method had a good inactivation effect on bovine viral diarrhea virus (BVDV) and pseudorabies virus (PRV), and had a certain inactivation effect on poliovirus Sabin type 1 (PV1).

[0169] Verification Example 3: Comparison of the effects of degassing membrane and direct introduction of CO into the solution

[0170] 50 mL of the pure hemoglobin prepared in Example 1 was taken and the degassing method of Example 1 was used for the operation; 50 mL of the pure hemoglobin prepared in Example 1 was taken and CO gas was directly introduced thereinto, and the introduction pipeline was placed at the bottom of the container. The ventilation flow rate of both methods was controlled at 40 mL / min, and samples were taken at intervals to detect the proportion of hemoglobin bound to CO. The results are shown in Table 5.

[0171] Table 5

[0172]

[0173] From the above results, when the two treatment methods were operated for 30 minutes, the ratio of hemoglobin bound to CO by the degassing method was close to 100%, while the ratio of hemoglobin bound to CO by the direct CO introduction method was 82.6%. Without considering the degassing method using the exhausted CO gas for recycling, the degassing method has a higher binding efficiency with hemoglobin when the gas consumption of both methods is 1.2L.

[0174] Verification Example 4: P50 Results of Different Groups

[0175] P50: refers to the oxygen partial pressure when the hemoglobin oxygen saturation is 50% (50% of the hemoglobin is bound to oxygen). That is, P50 reflects the ability of hemoglobin to bind and release oxygen. The higher the P50, the easier it is to release oxygen, and the lower the P50, the stronger the ability to bind oxygen.

[0176] Table 6

[0177]

[0178] Note: All samples were tested under the same experimental conditions.

[0179] The purpose of the formula design of the present invention is to maintain the P50 of the formula at a moderate value. Too high P50 is not conducive to hemoglobin binding to oxygen, so that hemoglobin binds less oxygen and the efficiency of transporting hemoglobin to the wound decreases; too low P50 is not conducive to the release of oxygen, and oxygen cannot be released quickly to the wound bed. Both of the above situations are not conducive to the healing of chronic wounds. Therefore, bovine hemoglobin is selected as the target protein. It can be seen from the above table that the P50 value of pure bovine hemoglobin is lower than the P50 value of polymerized bovine hemoglobin, but higher than the P50 of similar foreign products; after CO is introduced, the P50 of pure bovine hemoglobin and polymerized bovine hemoglobin is reduced. The P50 of pure bovine hemoglobin after binding with CO is higher than that of similar foreign products, which is in line with design expectations.

[0180] Verification Example 5: Antibacterial Effect Comparison Experiment

[0181] According to WS / T650-2019 "Antibacterial and Antibacterial Effect Evaluation Method", an antibacterial effect experiment was carried out. The wound dressing prepared in Example 1 and the energy-saving spray of Comparative Example 3 (similar products) were collected and contacted with different types of bacteria for 48 hours. The results are shown in Table 7.

[0182] Note: An antibacterial effect is considered when the inhibition rate is between 50% and 90%, and a strong antibacterial effect is considered when the inhibition rate is ≥90%.

[0183] From the results in Table 7, it can be seen that the wound dressing prepared in Example 1 has a strong inhibitory effect on Escherichia coli and Pseudomonas aeruginosa, while Comparative Example 3 has no inhibitory effect on the three bacterial species.

[0184] Table 7

[0185]

[0186] Verification Example 6: Real-time stability of products prepared in Examples and Control Examples

[0187] The prepared samples were placed in a refrigerator at 2-8°C for later use. About 1 mL of sample was drawn with a 1 mL syringe, and the content of different types of protein was detected with a blood gas analyzer. The results are shown in Table 8. The symbols represent the following meanings:

[0188] ctHb – total hemoglobin content;

[0189] sO2 – dissolved oxygen content in hemoglobin;

[0190] FO2Hb – the amount of hemoglobin bound to oxygen;

[0191] FCOHb – the amount of hemoglobin bound to CO;

[0192] FMetHb - methemoglobin content, reflecting the proportion of hemoglobin that has lost the ability to bind oxygen.

[0193] Table 8

[0194]

[0195] Example 1 is a product prepared by combining pure hemoglobin with CO. It can be seen that after the product was placed for 154 days after the formula filling, the content of methemoglobin was still low. After being placed for 190 days and sprayed in the air, part of the hemoglobin combined with oxygen in the air, the content of oxyhemoglobin increased by 10%, and the content of hemoglobin combined with CO decreased by nearly 10%. Although it was sprayed in the air, the content of methemoglobin was still less than 2%. This shows that the stability of the product has not decreased after being placed for more than half a year.

[0196] Example 2 is a product prepared by combining polymerized hemoglobin with CO. It can be seen that the content of CO combined with polymerized hemoglobin after the introduction of CO did not reach 100%, and the methemoglobin content of the product after 38 days of placement after formula filling was about 3%. The possible reason why the CO combination did not reach 100% is that hemoglobin is a tetramer, and the conformation of the protein after polymerization may hinder CO from combining with it.

[0197] Comparative Example 1 is a product prepared by combining pure hemoglobin without CO. It can be seen that the product after formula filling is placed for 15 days, and the methemoglobin content is relatively low, but the methemoglobin content in the product increases by more than 15% after 28 days, and part of the ability to bind oxygen is lost. This indicates that the stability of the product decreases after 28 days. Compared with the product prepared by combining pure hemoglobin with CO, it can be seen that CO has a good protective effect on hemoglobin.

[0198] Comparative Example 3 is the result of a similar product that has been marketed overseas and stored for 2 years, and the methemoglobin content is about 6%.

[0199] Verification Example 7: Comparison of protein conversion results of pure hemoglobin bound to CO and polymerized hemoglobin bound to CO under different environments

[0200] 7.1 Protein conversion experiment of pure hemoglobin after passing CO under different conditions

[0201] 20 mL of pure hemoglobin prepared in Comparative Example 1 was operated by degassing method, and samples were taken with a sterile syringe at 0, 60s, 90s, 120s, 150s, 180s, 210s, and 240s, respectively, and the protein content was detected by blood gas analyzer. The sample into which CO was introduced was placed in a glove box (with a high-purity nitrogen environment and extremely low oxygen content), and the protein content was detected by blood gas analyzer at regular intervals. The sample placed in the glove box for 84 days was placed in the air for a period of time, and the protein content was detected by blood gas analyzer at regular intervals. The results are shown in Table 9.

[0202] Table 9

[0203]

[0204] It can be seen from Table 9 that:

[0205] First, after passing CO for 3 minutes, the dissolved oxygen content continued to increase, and the oxygenated hemoglobin content showed an upward trend. Due to the synergistic effect, when one oxygen molecule binds to hemoglobin, it will accelerate the binding of other oxygen molecules to hemoglobin, so the oxygenated hemoglobin began to show an upward trend.

[0206] Second, continue to degas and introduce CO, the dissolved oxygen drops drastically to undetectable levels, and oxygenated hemoglobin begins to show a downward trend. This indicates that CO begins to compete with oxygen for binding to hemoglobin. Since the binding capacity of CO to hemoglobin is 300 times that of oxygen to hemoglobin, when the amount of CO in hemoglobin reaches a critical point, oxygen begins to separate from hemoglobin. Due to the synergistic effect of the tetramer, when one oxygen molecule is released from hemoglobin, it accelerates the rate of release of other oxygen molecules from hemoglobin. Therefore, the above phenomenon occurs, that is, after 210s of CO introduction, CO completely replaces O2 to bind to hemoglobin, and dissolved oxygen is discharged.

[0207] Third, the samples after CO was introduced were relatively stable when placed in a nitrogen environment. After being placed in a nitrogen environment for 84 days, the free hemoglobin (FHHb%) increased. This was the result of competition between nitrogen and CO, indicating that CO was debinding from hemoglobin.

[0208] Fourth, after the CO was introduced, the samples that had been placed in a nitrogen environment for 84 days were exposed to air for 3 hours. The content of hemoglobin bound to oxygen (FO2Hb) increased from 1.7% to about 30%, but methemoglobin was only oxidized by about 6%. The possible reason is that CO protects hemoglobin and prevents it from being oxidized to methemoglobin.

[0209] Fifth, when the sample after CO2 injection was placed in a nitrogen environment for 84 days and then exposed to air for 96 hours, the content of methemoglobin increased to 66.4%. At this time, there was still some hemoglobin that had not been oxidized into methemoglobin, and this part of hemoglobin still did not lose its activity in binding oxygen.

[0210] 7.2 Protein conversion experiment of polymerized hemoglobin after CO was introduced into different environments

[0211] 20 mL of polymerized hemoglobin prepared in Comparative Example 2 was operated by the degassing method of Example 1, and samples were taken with a sterile syringe at 0, 30s, 60s, 90s, 120s, 150s, 180s, 210s, 240s, 300s, and 600s, respectively, and the protein content was detected by a blood gas analyzer. The sample that was passed with CO for 600s was placed in a glove box (high-purity nitrogen environment), and the protein content was detected by a blood gas analyzer at regular intervals. The sample that was placed in the glove box for 78 days was placed in the air for a period of time, and the protein content was detected by a blood gas analyzer at regular intervals. The results are shown in Table 10.

[0212] Table 10

[0213]

[0214] As can be seen from Table 10, after CO was introduced into polymerized hemoglobin for 10 minutes, the content of CO-bound hemoglobin did not reach 100%, and the content of methemoglobin was about 4%. After being placed in a glove box (pure nitrogen environment) for 78 days, the content of methemoglobin was low. Due to the competition of nitrogen, CO and hemoglobin gradually decoupled, the content of free hemoglobin (FHHb) increased to 75.1%, and the content of CO-bound hemoglobin decreased to 23.7%. After polymerized hemoglobin was placed in a nitrogen environment for 78 days and placed in air for 3 hours, the content of oxygenated hemoglobin (FO2Hb) increased from 1.7% to nearly 30%, but the content of methemoglobin only increased by about 8%, which may be due to the role of CO in protecting hemoglobin from oxidation.

[0215] Compared with pure hemoglobin placed in air after CO was passed through it, the methemoglobin content in polymerized hemoglobin placed in air for 3 hours after CO was passed through it was higher than that in pure hemoglobin placed in air for 3 hours after CO was passed through it. This may be because the spatial structure of hemoglobin after polymerization hinders CO from completely binding with it.

[0216] Verification Example 8: Animal experiment on wound dressing prepared without CO

[0217] Diabetic foot ulcer is a serious complication of diabetes. It is a kind of chronic wound that is difficult to heal due to foot infection, ulcer or deep tissue damage caused by distal nerve abnormalities and vascular lesions in the lower limbs. More than 80% of diabetic foot ulcers are caused by type 2 diabetes. The modeling aims to establish a chronic wound model of diabetic skin ulcers in mice to simulate the chronic wounds formed by diabetic foot ulcers.

[0218] According to "YY / T1477.6-2020 Standard Test Model for Evaluation of Contact Wound Dressing Performance Part 6: Animal Type 2 Diabetes Refractory Wound Model for Evaluation of Wound Healing Performance", a model of type 2 diabetes skin ulcer mice was established. This experiment used knockout diabetic mice (db / db diabetic model mice) and established full-thickness skin excision wounds on their backs to simulate the occurrence and development of chronic wounds in type 2 diabetes.

[0219] The wound dressing prepared in Comparative Example 1 was applied to the modeled animals, and a marketed product was used as a positive control to evaluate the effect.

[0220] 1. Experimental methods

[0221] 1.1 Grouping

[0222] The 18 mice that passed the quarantine were tested for blood glucose and randomly divided into 3 groups according to their blood glucose levels, namely, a blank model group, a product group, and a positive control group (using the same product, i.e., Comparative Example 3). Each group contained 6 mice. The dressing of the product group was the dressing prepared in Comparative Example 1. The details are shown in Table 11.

[0223] Table 11

[0224]

[0225] 1.2 Modeling

[0226] Model construction: After depilation and anesthesia of the mouse back, a circular full-thickness skin tissue with a diameter of about 8 mm was cut off from the middle of the back to create a wound surface, apply glacial acetic acid, and fix the wound with a wound fixator.

[0227] 1.3 Grouping and processing methods are shown in Table 12.

[0228] Table 12

[0229]

[0230] Reason for selecting the amount of dressing applied: Based on the effective dose for other indications.

[0231] 1.4 Evaluation Nodes and Evaluation Indicators

[0232] Nodes: 0h, 1d, 3d, 7d, 15d;

[0233] Evaluation indicators: wound healing area and healing rate;

[0234] Wound healing rate = (initial wound area - wound area after treatment) / initial wound area × 100%.

[0235] 2. Results

[0236] 2.1 Wound healing area

[0237] In terms of wound area, compared with the saline group, the area ratio (wound area / inner circle area of ​​wound fixator) of the product group at 1d, 3d, 7d, and 15d after wound was consistent with that of the saline group; the wound area ratio of the positive drug group had a slower change trend than that of the saline group, and there was no statistical difference between the two groups of dressings and the saline group (p>0.05). The results are shown in Table 13.

[0238] Table 13

[0239]

[0240] 2.2 Wound healing rate

[0241] From the perspective of wound healing rate, compared with the saline group (blank model), the healing rate change trend of the product group was basically consistent with that of the saline group, and the healing rate change trend of the positive drug group was slower than that of the saline group. There was no statistical difference between the two dressing groups and the saline group (p>0.05). The results are shown in Table 14.

[0242] Table 14

[0243]

[0244] Note: The healing rates of the positive control groups D1 and D3 were negative because the wound was severely displaced 24 hours after the injury, resulting in an increase in the wound area. Therefore, the area measured on the second day was recorded as the initial wound area. The wound healing rate curve is shown in Figure 6 shown.

[0245] Based on the above results after treatment with the dressing, the wound area and cure rate of mice were compared with those of the blank model group. There was no statistical difference between the wound dressing prepared without CO and the control group and the model group.

[0246] Verification Example 9: Animal experiment on wound dressing prepared with CO

[0247] Compare the effects of the wound dressing prepared in Example 1 and Comparative Example 3 in treating chronic wound ulcers caused by type 2 diabetes.

[0248] 1. Experimental Methods

[0249] 1.1 Grouping

[0250] The 24 db / db mice that passed the quarantine were subjected to blood glucose test and randomly divided into 3 groups according to the blood glucose level, namely blank model group, product group, and positive control group (using the same product, i.e. comparative example 3), each group having 8 mice. The dressing of the product group was the dressing prepared in Example 1, as shown in Table 15.

[0251] Table 15

[0252]

[0253] 1.2 Modeling

[0254] All mice were anesthetized with 5% chloral hydrate at a dose of 8 mg / kg, and the backs were depilated. After the animal hair was trimmed with electric clippers, a layer of depilatory cream was applied on the skin surface. After 2 to 3 minutes, the depilatory cream was wiped off with a cotton swab, and the skin was cleaned with normal saline. Then a transparent fixing ring with an inner diameter of 12 mm was sutured and fixed to the central part of the back of the mouse. A skin sampler was used to gently press out a circular incision with a diameter of 10 mm in the fixing ring. Then surgical scissors were used to remove the full layer of skin along the incision, deep to the fascia layer, and normal saline was used for debridement after the operation.

[0255] 1.3 Grouping and processing methods are shown in Table 16.

[0256] Table 16

[0257]

[0258] Note: The excipient solution in the above table refers to the matrix solution that does not contain hemoglobin.

[0259] 1.4 Evaluation Nodes and Evaluation Indicators

[0260] Nodes: 0h, 1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, 17d, 19d, 21d, 23d.

[0261] Evaluation index: healing rate

[0262] Wound healing rate = (initial wound area - wound area after treatment) / initial wound area × 100%.

[0263] 2. Results

[0264] 2.1 Wound healing rate

[0265] The results are as follows Figure 7 As shown, from Figure 7 From the healing curve, the wound dressing prepared in Example 1 and the similar product of Comparative Example 3 were used to treat the type II diabetic skin ulcer model animals, and the healing rate at 23 days was better than that of the blank control group. Among them, on the 5th to 11th day, the healing rate of the positive control group was slightly higher than that of the experimental group. After the 11th day, the healing rate of the experimental group was higher than that of the positive control group, and there was a significant statistical difference compared with the blank model group. The possible reason is that the wound dressing prepared in the experimental group has a higher P50 value than the positive control group, and can release more oxygen to the wound in the later stage of the experiment, providing better healing conditions for the healing of diabetic ulcer wounds. At the same time, the antibacterial effect of Example 1 in the later stage of the experiment is also conducive to the inhibition of bacteria in chronic wounds, thereby accelerating the healing of wounds.

[0266] 2.2 Effect diagram of wound healing process

[0267] Figure 8 This is the wound healing effect diagram of the blank model group after 23 days. Fig. 9 This is a diagram showing the wound healing effect of the experimental group (Example 1) after 23 days of treatment. Fig.10 This is a diagram showing the wound healing effect of the positive control group (Comparative Example 3) after 23 days of treatment.

[0268] Verification Example 10: Animal experiment on wound dressings with different protein concentrations prepared by combining CO

[0269] The effects of the wound dressings with low, medium and high protein concentrations prepared according to Example 1 and Comparative Example 3 in treating chronic wound ulcers caused by type 2 diabetes were compared. The preparation method of the medium-concentration wound dressing is the method of Example 1. The preparation method of the high-concentration wound dressing is basically the same as that of Example 1, except that in step (V) of Example 1, the prepared auxiliary material solution is used to ultrafilter the inactivated hemoglobin through a 10kD membrane package, the liquid replacement multiple is maintained at 10 times, the membrane package transmembrane pressure during the liquid replacement process is 1.2 bar, and the hemoglobin solution after the liquid replacement is concentrated to 13 g / dL; the preparation method of the low-concentration wound dressing is also basically the same as that of Example 1, except that in step (V) of Example 1, the prepared auxiliary material solution is used to ultrafilter the inactivated hemoglobin through a 10kD membrane package, the liquid replacement multiple is maintained at 10 times, the membrane package transmembrane pressure during the liquid replacement process is 1.2 bar, the protein concentration in the feed liquid bottle is sampled and detected, and the feed liquid is diluted to 7 g / dL with the prepared auxiliary material solution according to the test result.

[0270] 1. Experimental Methods

[0271] 1.1 Grouping

[0272] The db / db mice that passed the quarantine were tested for blood glucose and randomly divided into 5 groups according to their blood glucose levels, namely, blank model group, low-dose group, medium-dose group, high-dose group, and positive control group (using similar products, i.e., comparative example 3), with 9 to 10 mice in each group. The low-dose, medium-dose, and high-dose groups used wound dressings with protein concentrations of 7 g / dL, 10 g / dL, and 13 g / dL, respectively. As shown in Table 17.

[0273] Table 17

[0274]

[0275] 1.2 Modeling

[0276] All mice were anesthetized with 2% isoflurane in 100% oxygen, and the backs were depilated, wound fixators were sutured, and full-thickness skin was excised with a circular wound of 10 mm in diameter.

[0277] 1.3 Grouping and processing methods are shown in Table 18.

[0278] Table 18

[0279]

[0280] Note: The excipient solution in the above table refers to the matrix solution that does not contain hemoglobin.

[0281] 1.4 Evaluation Nodes and Evaluation Indicators

[0282] Nodes: 0h, 1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, 17d, 19d, 21d, 23d, 25d, 27d, 29d.

[0283] Evaluation index: healing rate

[0284] Wound healing rate = (initial wound area - wound area after treatment) / initial wound area × 100%.

[0285] 2. Results

[0286] 2.1 Wound healing rate

[0287] The results are as follows Fig.11 As shown, from Fig.11 Judging from the healing curves, low-dose hemoglobin spray (7 g / dL) has a significant therapeutic effect on wound healing in diabetic ulcer mice.

[0288] 2.2 Effect diagram of wound healing process

[0289] The results are as follows Fig.12 As shown, from Fig.12 From the perspective of wound healing effect, the therapeutic effect of low-dose hemoglobin spray (7g / dL) on wound healing of diabetic ulcer mice lasted longer than that of the positive control group.

[0290] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a wound dressing, characterized in that: The preparation method comprises the following steps: (1) Preparation and preliminary purification of hemoglobin: Bovine blood is filtered through sodium citrate solution, then lysed with ultrapure water, and then filtered to obtain crude pure hemoglobin; the specific operation is as follows: The bovine blood is filtered through a capsule filter, the pressure at the liquid inlet end is controlled to be ≤4psi, the filtered liquid is collected, and the filtered liquid is washed with a sodium citrate solution, and the transmembrane pressure is maintained at ≤9psi during the washed filter. The liquid collected after the washed filter is lysed with ultrapure water, and the lysed liquid is passed through a 100kD hollow fiber membrane filtration system by a peristaltic pump, and the liquid at the permeate end of the membrane filtration system is collected and passed through a 10kD or 30kD hollow fiber column or membrane bag filtration system by a peristaltic pump, and the liquid at the reflux end is collected. When the conductivity of the collected liquid at the permeate end is ≤1ms / cm, the operation of the filtration system is stopped, and the liquid collected at the reflux end is passed through a 0.45μm and / or 0.22μm sterile filter to obtain crude pure hemoglobin; (2) Fine purification of hemoglobin: the crude pure hemoglobin is subjected to anion exchange chromatography treatment, and the collected chromatographic target eluate is further filtered to obtain finely purified hemoglobin; the specific operation is as follows: The crude pure hemoglobin is subjected to anion exchange chromatography treatment, the eluate before the elution peak is discarded, the middle peak is collected, the hemoglobin concentration is detected, and the collected chromatographic target eluate is passed through a 30kD membrane package or a hollow fiber column, and the liquid is replaced with a phosphate buffer at a constant volume, and the liquid replacement multiple is maintained at 7 to 10 times, and after completion, the pure hemoglobin is obtained; (3) Degassing: The purified hemoglobin is mixed with CO in reverse order to be degassed, and then sterilized and filtered to obtain degassed hemoglobin. The specific operation is as follows: The pure hemoglobin is collected, and the pure hemoglobin is pumped into the liquid inlet of the degassing device through a medical silicone pipeline by a peristaltic pump, and the liquid outlet is connected to the silicone pipeline, and the gas inlet of the degassing device opposite to the inlet of the liquid inlet is connected to a CO gas cylinder, and the gas outlet of the degassing device is connected to the gas inlet through a three-way pipeline. The purpose of such connection is to improve the utilization rate of CO so that the discharged CO gas can be recycled. The pure hemoglobin degassed by the degassing membrane is sterilized and filtered by 0.45+0.22μm, that is, sterilized and filtered by 0.45μm and 0.22μm membranes in sequence to obtain degassed hemoglobin; (4) Virus inactivation: The degassed hemoglobin is subjected to a virus inactivation process to obtain inactivated hemoglobin; the specific operation is as follows: The virus inactivation method is a low pH inactivation method or a chemical reagent inactivation method. The steps of the low pH inactivation method are as follows: adjusting the pH of degassed hemoglobin to 4.0±0.1 with a 100 mM hydrochloric acid solution, storing it in a refrigerator at 2-8° C. for 1 hour to 3 days, and detecting the protein content and P50; the steps of the chemical reagent inactivation method are as follows: adding 0.3% tributyl phosphate and 0.1%-0.5% sodium cholate to the degassed hemoglobin, placing it in a constant temperature incubator at 22-25° C. for 1 hour to 6 hours, detecting the protein content and P50, and obtaining inactivated hemoglobin; (5) Preparation of wound dressing stock solution: ultrafiltration of the auxiliary material and the inactivated hemoglobin to obtain the dressing stock solution; the specific operation is as follows: Prepare auxiliary materials and make the pH of auxiliary materials between 5.5 and 6.5; The prepared auxiliary material solution is ultrafiltered through a 10kD or 30kD membrane package to replace the inactivated hemoglobin, and the replacement multiple is maintained at 6-10 times. The hemoglobin solution after the replacement is concentrated, and the transmembrane pressure of the replacement and concentration steps is controlled at ≤1.5bar. The speed of the peristaltic pump during the replacement is controlled at ≤300r / min. The concentrated target protein solution is filtered through a sterile syringe filter into a pre-sterilized vial to obtain a wound dressing stock solution, and the protein content and P50 of the wound auxiliary material stock solution are tested. The stock solution after the test is stored at 2-8°C; (6) Preparation of wound dressing: Filling the dressing stock solution to obtain the wound dressing; the specific operation is as follows: Pour the wound dressing stock solution into the filling cup, control the volume not to exceed 20mL, physically connect the bottom of the filling cup with the valve of the inflated and sealed dual-package metal bottle, connect the filling cup with the downward pressure piston of the filling machine, the power source for driving the downward pressure piston is nitrogen, argon or helium inert gas, adjust the driving pressure to 0.6~0.75MPa, press the control piston rocker, and fill the stock solution in the cup into the dual-package metal bottle through the piston to obtain the wound dressing.

2. The method for preparing the wound dressing according to claim 1, characterized in that: In step (1), the sodium citrate solution is filtered and washed 8 to 16 times.

3. The method for preparing the wound dressing according to claim 1, characterized in that: In step (3), the flow rate of pure hemoglobin is 20-1000 mL / min; the CO introduction rate is 1-60 mL / min, and the CO introduction time is 1-210 min.

4. The method for preparing the wound dressing according to claim 1, characterized in that: In step (5), the auxiliary materials include antibacterial agents, antioxidants, electrolytes and pH regulators.

5. The method for preparing the wound dressing according to claim 4, characterized in that: The antibacterial agent includes one or a combination of two or more of benzyl alcohol, 1,2-hexanediol, and 2-phenoxyethanol; And / or, the antioxidant includes one or a combination of two or more of N-acetylcysteine, proanthocyanidins, cysteine, vitamin C, reduced glutathione, sodium sulfite, sodium dithionite, β-mercaptoethanol, dithiothreitol, and dithioerythritol.

6. The method for preparing the wound dressing according to claim 4, characterized in that: The electrolyte includes one or a combination of two or more of chloride ions, sodium ions, potassium ions, magnesium ions, and calcium ions; And / or, the pH adjuster includes one or a combination of two or more of sodium hydroxide, potassium hydroxide, disodium hydrogen phosphate, dipotassium hydrogen phosphate, and sodium dihydrogen phosphate.

7. The wound dressing obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the wound dressing according to claim 7 in the preparation of medical cosmetic products.

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