Microalgae wound patch based on temperature-sensitive phase change hydrogel

Abstract

The invention discloses a microalgae wound patch based on temperature-sensitive phase change hydrogel, and relates to the field of medical instruments. Comprising a sheet-shaped adhesive tape and a drug-containing absorption pad located above the middle of the sheet-shaped adhesive tape, the drug-containing absorption pad comprises a Pluronic F-127 layer, chlorella pyrenoidosa is evenly distributed in the Pluronic F-127 layer, and a sterile absorption isolation layer is arranged above the Pluronic F-127 layer. The temperature-sensitive phase-change hydrogel Pluronic is innovatively adopted as a microalgae culture medium in the patch, the oxygen production rate of algae can be increased, and the local oxygen concentration of a wound is increased. The Pluronic F-127 solution with the concentration of 25% can accelerate the oxygen production rate of chlorella, the high photosynthesis rate of microalgae is kept in the culture environment and the wound environment, and the production efficiency and the application effect are guaranteed. On the basis of the original related functions of hemostasis and disinfection of the wound patch, a stable uninterrupted oxygen supply function is added, and the effect of quickly healing the wound is achieved.

Description

Technical Field

[0001] The invention relates to the field of medical devices, and in particular to a microalgae wound patch based on a thermosensitive phase change hydrogel. Background Art

[0002] Band-aids are one of the most commonly used surgical drugs in our lives. They are mainly composed of dry adhesive tape, drug-containing absorbent pads and isolation permeable membranes. They have the function of temporarily stopping bleeding and protecting the wound surface. The most commonly used band-aid is the benzalkonium chloride patch, which contains a cationic surfactant. The drug-containing absorbent pad is in direct contact with the wound and has a certain bactericidal effect, but it cannot provide continuous oxygen therapy. In addition, under normal circumstances, the most vulnerable parts of the human body are the wrists, ankles, toes, noses, fingers and face. The epidermis of these parts is often not a typical flat structure, but a rugged surface composed of many depressions and protrusions. The drug-containing absorbent pad of a conventional band-aid that contacts the skin has a flat surface and often cannot fit the wound accurately, causing the wound to be polluted by the outside air and rubbed against the band-aid.

[0003] Microalgae is a general term for microorganisms that contain chlorophyll a and can perform photosynthesis, including prokaryotic algae (such as cyanobacteria) and eukaryotic algae (such as diatoms and green algae). They are widely found in oceans or freshwater lakes and are widely used in food, health products, and biofuels [1]. In recent years, researchers have gradually realized the great potential of microalgae as biomaterials in the medical field. Through photosynthesis, microalgae can continuously convert carbon dioxide into oxygen under light conditions to achieve a local oxygen supply effect, providing a source of active oxygen for treatment methods that are highly dependent on oxygen, such as radiotherapy and photodynamic therapy, thereby improving the treatment effect [2].

[0004] Wound healing is a process that is highly dependent on oxygen. Wound healing is a complex process that can be summarized into three stages: inflammatory phase, proliferative phase, and remodeling phase[3]. Oxygen is involved in each stage[4]. Repair activities such as cell proliferation, angiogenesis, and collagen synthesis also require oxygen. However, the common rupture or contraction of blood vessels in wounds hinders oxygen supply, leading to tissue hypoxia, which is not conducive to wound healing. Therefore, increasing the local oxygen concentration in the wound can effectively accelerate wound healing. The methods currently used in clinical practice, such as hyperbaric oxygen therapy or local gas oxygen therapy, have many problems such as poor sustainability of oxygen therapy, large energy loss, and insignificant effect. [5], and microalgae, as natural photosynthetic organisms, have unique advantages in accelerating wound healing. There are a large number of diabetic patients worldwide, and about 25% of diabetic patients are at risk of chronic wounds that do not heal throughout their lives [6]. Studies have confirmed that the delayed healing of chronic diabetic wounds is due to the damage to new blood vessels caused by hypoxia [7]. Chen et al. [8] developed a microalgae gel patch filled with gel beads containing active cyanobacteria, which can continuously deliver local dissolved oxygen to the wound in a humid environment, increase wound oxygenation, promote fibroblast proliferation and angiogenesis, and thus promote wound healing. In addition, Centeno-Cerdas et al. [9] Genetically modified Chlamydomonas reinhardtii were creatively implanted into commercially available surgical sutures to create photosynthetic sutures that can continuously release oxygen and recombinant human growth factor, effectively promoting wound healing.

[0005] At present, a major obstacle to the large-scale commercial use of microalgae in the biomedical field is the cultivation method of microalgae. Currently, large-scale cultivation modes of microalgae can be divided into two categories: open cultivation and closed cultivation. Among them, raceway pool cultivation and photobioreactor cultivation are the most common methods, and both are liquid suspension cultivation.

[10] . The runway pool has low operating costs and is conducive to scale expansion, but it is difficult to achieve precise control, making the microalgae easily contaminated or even completely worthless. Photobioreactor culture is finely controllable and highly repeatable, but it is expensive and must be equipped with cooling measures to prevent excessive temperatures in the system from affecting the growth of microalgae, making it difficult to expand. As for the application of microalgae in the biomedical field, especially wound healing, the bigger problem lies in the harvesting and reprocessing of microalgae. The manufacture of microalgae gel patches for wounds requires first harvesting the microalgae from the culture system before the microalgae can be processed. At this stage, the cost of harvesting microalgae from suspended culture systems is very high. Taking the production of biodiesel as an example, the cost incurred by the harvesting process can account for 20%-30% of the total cost of microalgae biodiesel.

[11] However, the application in the biomedical field has more stringent requirements on the pollution-free process of microalgae harvesting, which will lead to higher costs.

[0006] In this case, the solid phase culture method using gel as the culture medium has a good development prospect. First, using gel as the culture medium saves most of the post-harvest processing. Second, the harvest of solid phase culture is more efficient and energy-saving than suspension culture. Estime et al.

[12] A microalgae cultivation method that supports efficient harvesting was developed using thermosensitive phase change hydrogels. This method uses the thermal reversibility of thermosensitive phase change hydrogels to carry out efficient cultivation in the solid phase, cool it down to convert it into a liquid phase to allow the microalgae to settle, and then heat it up to fix it so that the microalgae at the bottom can be easily harvested. Pierobon et al.

[13] developed a gel cultivation method based on periodic harvesting. This method ensures the enrichment culture of the top layer of gel culture medium through top light sources, and then achieves periodic harvesting and the start of the next cycle of top layer enrichment by simply cutting off the top layer of culture medium.

[0007] Therefore, those skilled in the art are committed to preparing a microalgae wound patch based on a thermosensitive phase change hydrogel by culturing Chlorella vulgaris that grows stably and produces oxygen.

[0008] References

[0009] [1]GANGL D,ZEDLER JAZ,RAJAKUMAR PD,et al.Biotechnologicalexploitation of microalgae[J].J Exp Bot,2015,66(22):6975-6990.

[0010] [2] QIAO Y, YANG F, XIE T, et al. Engineered algae: a novel oxygen-generating system for effective treatment of hypoxic cancer [J]. Sci Adv, 2020, 6(21): eaba5996.

[0011] [3]HUNT TK, BURKE J, BARBUL A, et al. Wound healing[J]. Science, 1999, 284(5421):1775.

[0012] [4]BROUGHTON G,JANIS JE,ATTINGER C E.The basic science ofwoundhealing[J].Plast Reconstr Surg,2006,117(7Suppl):12S-34S.

[0013] [5]HEYBOER M, SHARMA D, SANTIAGO W, et al. Hyperbaric oxygen therapy: sideeffects defined and quantified[J]. Adv Wound Care, 2017, 6(6): 210-224.

[0014] [6]HART T,MILNER R,CIFU A.Management of a diabetic foot[J].JAMA, 2017, 318(14):1387-1388.

[0015] [7]GONZALEZ FJ,XIE C,JIANG C.The role of hypoxia-inducible factors in metabolic diseases[J].Nat Rev Endocrinol,2019,15(1):21-32.

[0016] [8]CHEN H, CHENG Y, TIAN J, et al. Dissolved oxygen from microalgae-gelpatch promotes chronic wound healing in diabetes[J]. Sci Adv, 2020, 6(20):eaba4311.

[0017] [9]CENTENO-CERDAS C, -CORDERO M, MN,et al.Development of photosynthetic sutures for the local delivery of oxygen and recombinantgrowth factors in wounds[J].Acta Biomater,2018,81:184-194.

[0018]

[10] Han Yanxia. Establishment and optimization of Spirulina float culture technology[D]. Hebei Agricultural University, 2022. DOI: 10.27109 / d.cnki.ghbnu.2022.000620.

[0019]

[11] Wu Penghui, Liu Yao, Zhang Lei, et al. Research progress on two-stage cultivation technology of microalgae [J / OL]. Bulletin of Microbiology: 1-15 [2023-09-21]. DOI: 10.13344 / j.microbiol.china.230471.

[0020]

[12] ESTIME B,REN D,SURESHKUMAR R.Cultivation and energy efficient harvesting ofmicroalgae using thermoreversible sol-gel transition[J].Scientific Reports,2017,7.

[0021] PIEROBON SC,RIORDON J,NGUYEN B,et al.Periodic Harvesting ofMicroalgae From Calcium Alginate Hydrogels for Sustained High-DensityProduction[J].Biotechnology and Bioengineering,2017,114(9):2023-31. Summary of the invention

[0022] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to prepare a microalgae wound patch based on a thermosensitive phase change hydrogel by culturing Chlorella vulgaris that grows stably and produces oxygen.

[0023] To achieve the above object, the present invention provides the use of Pluronic F-127 in preparing a wound patch carrier.

[0024] In a preferred embodiment of the present invention, the mass fraction concentration of Pluronic F-127 is 20%-30%.

[0025] Preferably, the mass fraction concentration of Pluronic F-127 is 25%.

[0026] The present invention also provides a microalgae wound patch based on a thermosensitive phase change hydrogel, comprising a sheet-like tape and a drug-containing absorption pad located above the middle of the sheet-like tape, wherein the drug-containing absorption pad comprises a Pluronic F-127 layer, wherein Chlorella vulgaris is evenly distributed in the Pluronic F-127 layer, and a sterile absorption isolation layer is arranged above the Pluronic F-127 layer.

[0027] In a preferred embodiment of the present invention, the sheet-like tape is composed of a backing material and an adhesive on the backing material, and the Pluronic F-127 layer is located between the adhesive and the sterile absorbent isolation layer.

[0028] Furthermore, the adhesive is selected from any one of rubber adhesive, medical hot melt adhesive, and solvent adhesive.

[0029] Furthermore, the backing material is a transparent structure.

[0030] In a preferred embodiment of the present invention, the sterile absorbent isolation layer is made of a highly water-absorbent material.

[0031] Preferably, the highly water-absorbent material is cotton.

[0032] In a preferred embodiment of the present invention, the method for uniformly distributing pyrenoid chlorella in the Pluronic F-127 layer is as follows: Pluronic F-127 is added to ultrapure water, stirred until completely dissolved, and then pyrenoid chlorella culture medium is added, and then the pyrenoid chlorella suspension is inoculated for cultivation.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention innovatively uses Chlorella pyrenoidosa to continuously release oxygen through photosynthesis, and the Chlorella is always in the same medium as the culture environment, ensuring the oxygen release rate. The backing material of the sheet tape is a transparent structure that can continuously absorb external light to maintain photosynthesis, and the innovative use of temperature-sensitive phase change hydrogel Pluronic as the microalgae culture medium in the patch can accelerate the oxygen production rate of algae and increase the local oxygen concentration in the wound. Pluronic F-127 solution at a concentration of 25% can accelerate the oxygen production rate of Chlorella, maintain a high photosynthesis rate of microalgae in both the culture and wound environments, and ensure production efficiency and application effects. On the basis of the original hemostatic and disinfection-related functions of the wound patch, a stable and uninterrupted supply function is added to achieve the effect of accelerating wound healing.

[0035] The present invention innovatively uses Pluronic F-127 material. With its thermoreversible properties, it is liquid at room temperature. When covered on the body surface (at about 32°C), it can solidify into an adaptive shape according to the specific shape of the skin and accurately fit the skin wound, thus achieving liquid preservation at room temperature and shaping effect after fitting the skin.

[0036] The present invention adopts liquid culture of 25% concentration of Pluronic F-127 at room temperature, which can increase the fluorescence value of Chlorella to more than twice that of 0% concentration, greatly improve the growth and oxygen release rate of Chlorella, accelerate the culture rate of Pyrenoid Chlorella and still achieve stable growth and oxygen production after solidification at 32°C.

[0037] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A front structural diagram of a microalgae wound patch based on a thermosensitive phase change hydrogel provided in an embodiment of the present invention;

[0039] Figure 2 A side structural diagram of a microalgae wound patch based on a thermosensitive phase change hydrogel provided in an embodiment of the present invention;

[0040] In the figure: 1, medicated absorbent pad; 1a, Chlorella pyrenoidosa; 1b, sterile absorbent isolation layer; 1c, 25% Pluronic F-127 layer; 2 sheet tape; 2a, adhesive of sheet tape; 2b, backing material of sheet tape;

[0041] Figure 3 The chlorophyll fluorescence values ​​of Chlorella pyrenoidosa after two weeks of cultivation at different pH salt concentrations;

[0042] Figure 4 The RFU values ​​are at room temperature and 32°C when inoculated, cultured for one week, and cultured for two weeks;

[0043] Figure 5 This is the PAM measurement data at room temperature and 32°C. DETAILED DESCRIPTION

[0044] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0045] In the drawings, components with the same structure are indicated by the same numerical reference numerals, and components with similar structures or functions are indicated by similar numerical reference numerals. The size and thickness of each component shown in the drawings are arbitrarily shown, and the present invention does not limit the size and thickness of each component. In order to make the illustration clearer, the thickness of the components is appropriately exaggerated in some places in the drawings.

[0046] Example 1

[0047] like Figure 1 , Figure 2 The microalgae wound patch based on the thermosensitive phase change hydrogel shown is composed of two parts: a drug-containing absorption pad 1 and a back layer sheet-like adhesive tape 2.

[0048] The medicated absorption pad 1 is the core part of the wound patch, and comprises a Pluronic F-127 layer, Chlorella pyrenoidosa 1a and a sterile absorption isolation layer 1b.

[0049] Pluronic is an amphiphilic ABA copolymer consisting of a hydrophobic polypropylene oxide (PPO, B) block and a hydrophilic polyethylene oxide (PEO, A) block. Pluronic F-127 has good biocompatibility and safety, and improves the growth rate and oxygen release rate of Chlorella.

[0050] In the present invention, the sterile absorption isolation layer 1b is made of cotton or other highly absorbent materials. The sterile absorption isolation layer 1b is in direct contact with the wound, and its main function is to absorb blood and other body fluids secreted by the wound, keep the wound dry, and thus promote wound healing. The pyrenoid chlorella 1a is evenly distributed in the 25% Pluronic F-127 layer 1c, and the 25% Pluronic F-127 layer 1c is located between the sterile absorption isolation layer 1b and the sheet tape 2, which can solidify into a fixed shape during the use of the wound patch and play a role in continuous oxygen release.

[0051] The sheet tape 2 consists of a backing material 2b and an adhesive 2a on the backing material 2b. The Pluronic F-127 layer is located between the adhesive and the sterile absorbent isolation layer. The adhesive 2a of the sheet tape is generally a rubber adhesive (natural rubber), a medical hot melt adhesive (synthetic rubber), a solvent adhesive (acrylic acid), etc. The backing material 2b of the sheet tape is a transparent structure, and there are eight types, including elastic fabric, non-elastic fabric, PE film, PVC film, non-woven fabric, foam film, PU film, POE film, etc.

[0052] Example 2

[0053] Evenly distribute pyrenoid Chlorella in the Pluronic F-127 layer: Add Pluronic F-127 (hereinafter referred to as p-salt) to ultrapure water, stir vigorously with a magnetic stirrer until completely dissolved (ice bath stirring is required when the p-salt concentration is high), then add commercial BG-11 liquid culture medium, the ingredients of which are shown in Table 1, stir until completely dissolved, and then inoculate the pyrenoid Chlorella suspension for cultivation.

[0054] Table 1 BG-11 liquid culture medium composition

[0055]

[0056]

[0057] Experimental Example 1: Investigating the Effect of P Salt Concentration on Its Phase Transition Point

[0058] Pluronic has good biocompatibility, biosafety and thermal reversibility. When heated above the gelation temperature Tg, the Pluronic aqueous solution undergoes a phase change and becomes an elastic gel. By cooling the gel below Tg, the gel can return to the liquid phase.

[0059] The thermal reversibility of p-salt is the basis of the present invention. When inoculating Chlorella pyrenoidosa, the culture system should be in the liquid phase; at the working temperature (wound environment) of about 32°C, the system should be in the solid phase. Therefore, it is necessary to find a suitable p-salt concentration so that the phase transition point of the system is between the two.

[0060] By adjusting the concentration of the multipolymer of the Pluronic F-127 material in the water phase, it was found that when the p-salt concentration was 10% or less, no phase transition was observed below 35°C. When the p-salt concentration was 15%, the phase transition point was observed to be about 25-32°C, which can achieve liquid storage at room temperature and shaping effect after skin adhesion.

[0061] Experimental Example 2: Investigation of continuous cultivation of Chlorella at different pH concentrations

[0062] The ability to support efficient cultivation is a major basis for the application of p-salts. Therefore, it is necessary to find the p-salt concentration that gives Chlorella the highest growth rate under room temperature cultivation conditions.

[0063] The present invention prepares two portions of liquid culture medium with β-salt concentrations of 0%, 10%, 15%, 20%, 25%, and 30%, each portion of 50 ml, inoculates an equal amount of pyrenoid chlorella suspension, and determines the initial chlorophyll fluorescence value to be 3444.32. The chlorophyll fluorescence value is measured after one week and two weeks of continuous culture. The results are shown in FIG. Figure 3 It can be seen that 20%, 25%, and 30% p-salt concentrations have obvious advantages in the growth rate of continuously cultured Chlorella, among which 25% p-salt concentration is the best.

[0064] Experimental Example 3: Investigation of continuous cultivation of Chlorella at different temperatures

[0065] Ultimately, the wound gel microalgae patch needs to work in a wound environment, so the growth rate of Chlorella in the culture system at different temperatures needs to be verified.

[0066] The present invention selects a 25% p-salt concentration, prepares 4 50 ml culture media, inoculates the same amount of Chlorella suspension, and measures the initial chlorophyll fluorescence value to be 10451.07. Two of the culture media are placed at room temperature, and two of the culture media are placed in a 32°C water bath for heating to simulate the wound environment. The results are shown in FIG. Figure 4 .

[0067] The growth rate of 32℃ culture was significantly higher than that of room temperature in the first week, but the growth rate at room temperature became significantly faster in the second week. The experiment proved that Chlorella can grow efficiently at room temperature with 25% p-salt, and can also grow normally in the wound environment, verifying the feasibility of p-salt as a wound patch carrier.

[0068] Experimental Example 4: Investigating the photosynthetic efficiency of Chlorella at different temperatures

[0069] Ultimately, the wound gel microalgae patch needs to work in a wound environment, so it is necessary to verify the photosynthetic efficiency of Chlorella in the culture system at different temperatures.

[0070] The present invention prepares two portions of 100 ml of 25% p-salt solution, adds 0.2 g of pyrenoid chlorella culture medium, and adds 80 ml of chlorella suspension. All are cultured at room temperature for one week, and then cultured at room temperature and 32°C for one week respectively, and then measured using PAM. The results are shown in Table 2 and Figure 5 .

[0071] Table 2 PAM determination data at room temperature and 32°C

[0072]

[0073]

[0074] From the above, we can see that the maximum photosynthetic capacity and actual photosynthetic rate of Chlorella at 32°C are only slightly lower than those at room temperature, and it can complete normal physiological and biochemical processes. This proves that the P-salt-based culture system can basically guarantee the oxygen release effect of Chlorella in the wound environment, verifying the feasibility of the wound microalgae patch.

[0075] In summary, according to the above experimental data, the present invention selects 25% as a more reasonable p-salt concentration. At this concentration, the culture system is in a liquid phase at room temperature and has viscosity. This makes the culture have the high efficiency of gel solid phase culture, and in theory, it can also be applied to the photobioreactor used in the current mainstream microalgae culture, indicating that the commercial application of this culture system has a bright future.

[0076] In addition, continuous culture experiments at room temperature confirmed that the culture efficiency of this culture system is better than that of traditional suspension culture, and the gel solid phase culture system is simple and energy-saving in the harvesting operation process. The final PAM measurement results showed that the photosynthetic rate of Chlorella vulgaris in the wound environment under this culture system can meet the requirements of oxygen release rate.

[0077] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. Application of Pluronic F-127 in the preparation of wound patch carriers.

2. The use according to claim 1, characterized in that: The mass fraction concentration of the Pluronic F-127 is 20%-30%.

3. The use according to claim 2, characterized in that: The mass fraction concentration of Pluronic F-127 is 25%.

4. A microalgae wound patch based on a thermosensitive phase change hydrogel, characterized in that: The invention comprises a sheet-shaped adhesive tape and a drug-containing absorption pad located above the middle of the sheet-shaped adhesive tape. The drug-containing absorption pad comprises a Pluronic F-127 layer, in which pyrenoid chlorella is evenly distributed, and a sterile absorption isolation layer is arranged above the Pluronic F-127 layer.

5. The microalgae wound patch based on thermosensitive phase change hydrogel according to claim 4, characterized in that: The sheet-like adhesive tape consists of a backing material and an adhesive on the backing material, and the Pluronic F-127 layer is located between the adhesive and the sterile absorbent isolation layer.

6. The microalgae wound patch based on thermosensitive phase change hydrogel according to claim 5, characterized in that: The adhesive is selected from any one of rubber adhesive, medical hot melt adhesive and solvent adhesive.

7. The microalgae wound patch based on thermosensitive phase change hydrogel according to claim 5, characterized in that: The backing material is a transparent structure.

8. The microalgae wound patch based on thermosensitive phase change hydrogel according to claim 4, characterized in that: The sterile absorbent isolation layer is made of highly water-absorbent material.

9. The microalgae wound patch based on thermosensitive phase change hydrogel according to claim 8, characterized in that: The highly water-absorbent material is cotton.

10. The microalgae wound patch based on thermosensitive phase change hydrogel according to claim 4, characterized in that: The method for uniformly distributing pyrenoid algae in the Pluronic F-127 layer is as follows: Pluronic F-127 is added into ultrapure water, stirred until completely dissolved, and then pyrenoid algae culture medium is added, and then the pyrenoid algae suspension is inoculated for cultivation.

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