Sustained-release microneedle, preparation method and application of sustained-release microneedle in preparation of drugs or instruments for treating septicopyemia
By preparing hydrogel sustained-release microneedles loaded with GCA protein, the problems of limited efficacy of sepsis drugs and difficulty in maintaining the active protein structure in the prior art are solved, and skin-released GCA proteins are achieved to treat sepsis through the treatment of sepsis, improving the treatment effect and patient compliance.
Patent Information
- Application Number
- CN202510525086.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-24
- Publication Date
- 2025-08-15
AI Technical Summary
The prior art has limited efficacy, safety issues, and lack of targeted immune regulation drugs in the treatment of sepsis, and the structure of active proteins is difficult to maintain when preparing hydrogel sustained release microneedles, resulting in poor treatment effect.
Hydrogels were prepared by cross-polymerization of low-temperature-sensitive methacrylated gelatin and acrylated polyethylene glycol NHS ester cross-polymerization. The sustained-release microneedle loaded with particulate calcin (GCA) was used to slowly release GCA protein through the skin to achieve immune regulation.
Effective treatment of sepsis was achieved, pain and discomfort were reduced, patient compliance was improved, and the pharmacological effect of GCA protein was similar to that of intravenous injection and had high structural stability.
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Figure CN120478259A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedical technology and relates to hydrogel sustained-release microneedles and their preparation method and application, and specifically to a sustained-release microneedle, its preparation method and application in the preparation of drugs or devices for treating sepsis. Background Art
[0002] Globally, more than 19 million patients develop sepsis each year, with a mortality rate as high as 30%. Among patients with septic shock, the mortality rate can reach 80%. Sepsis is a leading cause of death in intensive care units (ICUs), and the risk is particularly high among those with underlying medical conditions (such as cirrhosis and diabetes) or those with immunosuppression.
[0003] Currently, clinical treatments for sepsis primarily include broad-spectrum antibiotics, fluid resuscitation, and vasoactive drugs. Although these drugs can improve sepsis to a certain extent, they still present challenges in terms of efficacy and safety. For example, long-term use of broad-spectrum antibiotics may lead to increased bacterial resistance and reduced efficacy (especially for multidrug-resistant bacteria); fluid resuscitation is generally ineffective and can easily lead to excessive fluid load, causing complications such as pulmonary edema. Vasoactive drugs may cause arrhythmias and poor limb perfusion. Immune dysregulation is the core pathology of sepsis, but no targeted immunomodulatory drugs are currently widely used; various immunotherapies (such as IL-6 inhibitors and GM-CSF) are still in clinical trials. Therefore, there is an urgent need for a drug to improve the immune function of patients with sepsis.
[0004] Based on the applicant's discovery, the present invention is proposed. Summary of the Invention
[0005] The present invention aims to overcome the shortcomings of the existing technology. The first purpose is to provide a hydrogel sustained-release microneedle loaded with particulate calcitonin (GCA protein), the second purpose is to provide a preparation method of the hydrogel sustained-release microneedle loaded with GCA protein, and the third purpose is to provide the use of the hydrogel sustained-release microneedle loaded with GCA protein in the preparation of drugs or devices for treating sepsis.
[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:
[0007] A hydrogel sustained-release microneedle loaded with granular calcineurin, the hydrogel sustained-release microneedle comprising:
[0008] Base substrate: The base substrate has a microneedle side and an opposing back side;
[0009] Solid microneedle: The solid microneedle extends from the microneedle side of the base substrate and includes a hydrogel polymer and granular calcium protein, wherein: the hydrogel polymer is obtained by cross-linking polymerization of low-temperature sensitive methacrylated gelatin and acrylated polyethylene glycol NHS ester with a photoinitiator, and the granular calcium protein is dispersed in the hydrogel polymer.
[0010] Preferably, based on the low-temperature sensitive methacrylated gelatin, 20 μg to 25 μg of acrylated polyethylene glycol NHS ester is used for every 1.5 g of the low-temperature sensitive methacrylated gelatin.
[0011] Preferably, based on the low-temperature sensitive methacryloyl gelatin, 1 mg to 1.2 mg of granular calcineurin is used for every 1.5 g of the low-temperature sensitive methacryloyl gelatin.
[0012] Preferably, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphate.
[0013] More preferably, based on the low-temperature sensitive methacrylated gelatin, 0.03 g to 0.04 g of lithium phenyl-2,4,6-trimethylbenzoyl phosphate is used per 1.5 g of the low-temperature sensitive methacrylated gelatin.
[0014] Preferably, the cross-linking polymerization conditions are: irradiation curing at 405 nm to 410 nm for 30 s to 35 s.
[0015] Preferably, the thickness of the base substrate is 600 to 700 microns, and the length of the solid microneedles is 550 to 650 microns.
[0016] A method for preparing the above-mentioned hydrogel sustained-release microneedles comprises the following steps:
[0017] preparing a buffer solution containing a photoinitiator;
[0018] dissolving the low-temperature sensitive methacrylated gelatin with a portion of the buffer solution to obtain a first mixed solution;
[0019] adding granular calcein and acryloyl-polyethylene glycol NHS ester to the remaining buffer solution to obtain a second mixed solution;
[0020] The first mixed solution and the second mixed solution are mixed, added into a microneedle mold, concentrated, cross-linked and solidified, dried, and demoulded to obtain the microneedle mold.
[0021] Preferably, the buffer solution is phosphate buffer.
[0022] Application of the above hydrogel sustained-release microneedles in the preparation of drugs or devices for treating sepsis.
[0023] Beneficial effects:
[0024] 1. The present invention provides a hydrogel sustained-release microneedle loaded with GCA protein, which can slowly release GCA protein through the skin and has a significant therapeutic effect on septic mice. Those skilled in the art know that the maintenance of the higher-order structure of the active protein is crucial for it to exert its pharmacological effects. At the same time, the higher-order structure of the active protein is very sensitive to changes in external physical and chemical properties. When the active protein is loaded on the hydrogel sustained-release microneedle, the presence of organic solvents and the cross-linking reaction between organic substances make the maintenance of the higher-order structure of the active protein extremely uncertain. This is the biggest challenge when using active proteins to prepare hydrogel sustained-release microneedles, and often leads to failure. The hydrogel sustained-release microneedle loaded with GCA protein of the present invention can not only slowly release the GCA protein cross-linked therein, but also the released GCA protein still has excellent pharmacological effects, and there is no significant difference in the therapeutic effect with the injection of GCA protein.
[0025] 2. Compared with intravenous injection, the GCA protein-loaded hydrogel sustained-release microneedles provided by the present invention can reduce the pain and discomfort of use, and are more convenient to store and carry, thereby improving patient compliance. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 The flowchart of the construction of GCA@NHS / GelMA-MNPs and the finished product of GCA@NHS / GelMA-MNPs;
[0027] Figure 2 The structure of GCA@NHS / GelMA-MNPs under SEM electron microscope and its penetration force diagram;
[0028] Figure 3 The effects of GCA@NHS / GelMA-MNPs with different concentrations of GCA on the survival period of septic mice and the levels of inflammatory factors (IL-1β, IL-6 and TNF-α) in the serum;
[0029] Figure 4 GCA release rate diagram of GCA@NHS / GelMA-MNPs in vitro (A) and in vivo (B);
[0030] Figure 5 The effects of GCA@NHS / GelMA-MNPs and PBS@NHS / GelMA-MNPs on the survival period (A), body surface temperature (B) and anal temperature (C) of septic mice are shown;
[0031] Figure 6 Effects of GCA@NHS / GelMA-MNPs and PBS@NHS / GelMA-MNPs on liver and lung injury in septic mice;
[0032] Figure 7Effects of GCA@NHS / GelMA-MNPs and PBS@NHS / GelMA-MNPs on the bacterial population in the liver of septic mice;
[0033] Figure 8 Comparison of the therapeutic effects of tail vein administration of GCA and GCA@NHS / GelMA-MNPs in treating septic mice;
[0034] Figure 9 Effects of GCA@NHS / GelMA-MNPs and PBS@NHS / GelMA-MNPs on the phagocytic ability of bone marrow-derived macrophages in septic mice. DETAILED DESCRIPTION
[0035] The essential contents of the present invention are described in detail below with reference to the embodiments, but the protection scope of the present invention is not limited thereto.
[0036] Grancalcin (GCA) is a calcium-binding protein that belongs to the penta-EF-hand (PEF) protein family and contains five EF-hand motifs. It is primarily secreted by myeloid immune cells such as neutrophils and macrophages (primarily pro-inflammatory M1 macrophages). The GCA used in this invention is a conventional commercial GCA protein. All other reagents used in this invention, unless otherwise specified, are conventional commercial reagents.
[0037] Example 1: Construction of GCA@NHS / GelMA-MNPs
[0038] 0.03 g of photoinitiator lithium phenyl-2,4,6-trimethylbenzoyl phosphate (LAP) was added to 10 ml of phosphate buffer (0.01 M), heated in a 40-50° C. water bath to dissolve for 15 min, and shaken once every 3 min to obtain solution A.
[0039] Solution A was divided equally into two portions. 1.5 g of low-temperature-sensitive methacryloyl gelatin (WT-GelMA) was added to one portion and dissolved in a 40-50°C water bath for 50 min, with shaking every 10 min to obtain Solution B. 1 mg of GCA protein and 20 μg of acrylated polyethylene glycol NHS ester (NHS) were added to the other portion. After dissolution at 37°C, the mixture was incubated at 4°C for 18-24 h to obtain Solution C.
[0040] Solution B and solution C were sterile filtered, returned to 37°C, and mixed to obtain a mixed solution D.
[0041] The mixed solution D was added to the microneedle mold and centrifuged at 3000 rpm for 5 min 5-9 times to remove bubbles until the bubbles disappeared.
[0042] The mixture was concentrated in an oven at 37°C for 6-7 h, cured with a UV lamp (405 nm) for 30-32 s, dried in an oven at 37°C for 14-19 h, and demolded to obtain GCA@NHS / GelMA-MNPs microneedles.
[0043] The construction process of the above GCA@NHS / GelMA-MNPs is as follows Figure 1 As shown in A, the finished product of GCA@NHS / GelMA-MNPs is shown in Figure 1 As shown in B. Figure 1 In Figure B, it can be observed that the prepared microneedle patch is square and the microneedles are evenly arranged with consistent needle shape.
[0044] Example 2: Mechanical properties of GCA@NHS / GelMA-MNPs
[0045] This example is to measure the mechanical properties of the microneedles prepared in Example 1, as follows:
[0046] After vacuum freeze-drying, GCA@NHS / GelMA-MNPs were used to photograph their structure using a SEM electron microscope; a universal testing machine was used to test the microneedle puncture force; and the changes in the needle holes in the mouse skin were observed after GCA@NHS / GelMA-MNPs were used on mice. Figure 2 In Figure A, it can be seen that the surface of the GCA@NHS / GelMA-MNPs microneedles is smooth and needle-like, with a good porous structure; Figure 2 Middle B shows that the microneedle puncture force is significantly higher than 0.5N / needle, meeting the mechanical requirements for puncturing the skin; Figure 2 Figure C in the middle shows that GCA@NHS / GelMA-MNPs can penetrate the skin, and the wound will recover in about 60 minutes. The above results show that GCA@NHS / GelMA-MNPs can penetrate the skin and cause little trauma to the skin.
[0047] Example 3: Screening of GCA concentration in GCA@NHS / GelMA-MNPs
[0048] This example is to screen the optimal concentration of GCA protein for preparing microneedles, as follows:
[0049] In order to clarify the optimal concentration of GCA in GCA@NHS / GelMA-MNPs, the present invention constructed GCA@NHS / GelMA-MNPs with GCA contents of 0, 50, 100, and 200 μg / ml, and used microneedles of different concentrations to treat sepsis model mice. The survival period of the mice was recorded, and the levels of inflammatory factors (IL-1β, IL-6, and TNF-α) in the serum were detected using an Elisa kit. The results are shown in Figure 2. Figure 3 As shown, Figure 3Figure A in the middle shows that when the GCA concentration in the microneedle patch is greater than or equal to 100 μg / ml, the survival period of septic mice can be maximized; Figure 3 The BD in the middle showed that when the GCA concentration was greater than or equal to 100 μg / ml, the inflammation level in septic mice could be better controlled.
[0050] Example 4: In vitro and in vivo pharmacokinetic results of GCA@NHS / GelMA-MNPs
[0051] This example is to determine the pharmacokinetic characteristics of the microneedles prepared in Example 1, as follows:
[0052] In vitro: GCA@NHS / GelMA-MNPs were placed in sufficient PBS, and the GCA protein content in the PBS solution was detected by Elisa kit at 4, 8, 16, 24, and 32 hours.
[0053] In vivo: GCA@NHS / GelMA-MNPs were used to treat septic mice. Blood was collected from the tail vein of the mice at 0, 12, 24, 36, 48, and 72 hours, and the level of GCA protein in the serum was detected using an ELISA kit.
[0054] from Figure 4 In Figure A, it can be found that after 16 hours in vitro, the release rate of GCA@NHS / GelMA-MNPs slowed down significantly and basically reached its peak at 24 hours.
[0055] Figure 4 The results in middle B showed that after septic mice used GCA@NHS / GelMA-MNPs, the concentration in the body reached the highest between 36 and 48 hours, and the GCA concentration in the body gradually decreased after 48 hours.
[0056] Example 5: Therapeutic effect of GCA@NHS / GelMA-MNPs on septic mice
[0057] This example was designed to determine the therapeutic effect of the microneedles prepared in Example 1 on mice with sepsis. A comparative sample, PBS@NHS / GelMA-MNPs, was prepared using the same method as the GCA@NHS / GelMA-MNPs in Example 1, except that an equal mass of PBS was used in place of GCA. The therapeutic effect was determined as follows:
[0058] Mice with sepsis were randomly divided into a control group and a GCA@NHS / GelMA-MNPs-treated group. The control group was treated with PBS@NHS / GelMA-MNPs, while the treatment group was treated with GCA@NHS / GelMA-MNPs every three days. The mice's survival period was recorded, and their surface temperature was measured using a surface thermometer and their rectal temperature was measured using a rectal thermometer.
[0059] The results are as follows Figure 5 As shown in Figure 2, compared with the control group, the GCA@NHS / GelMA-MNPs treatment group significantly improved the survival period of septic mice ( Figure 5 Middle A), increase the body surface of mice ( Figure 5 Middle B) and rectal temperature ( Figure 5 (C) After three days, the mice in each group were killed and the liver and lung tissues were collected. The liver and lung tissues on one side were fixed with formaldehyde, embedded in paraffin, and sectioned. After staining with hematoxylin-eosin (HE), the tissue damage results were observed using an optical microscope. Figure 6 As shown in Figure 2, GCA@NHS / GelMA-MNPs can alleviate liver and lung damage in septic mice. The liver tissue was ground and filtered, and the bacterial content of the liver tissue was detected by plate colony counting method. The results are shown in Figure 2. Figure 7 As shown in the figure, it shows that after treatment with GCA@NHS / GelMA-MNPs, the bacterial content in septic mice was significantly reduced. The above results all indicate that GCA@NHS / GelMA-MNPs has a good therapeutic effect on septic mice.
[0060] Example 6: Comparison of the therapeutic effects of GCA@NHS / GelMA-MNPs and GCA on septic mice
[0061] This example is to clarify whether the administration of GCA@NHS / GelMA-MNPs prepared in Example 1 affects the therapeutic effect of the GCA protein itself, as follows:
[0062] In this example, septic mice were randomly divided into three groups. The control group was treated with PBS, the treatment group 1 was treated with GCA@NHS / GelMA-MNPs once every three days, and the treatment group 2 was treated with intravenous injection of GCA (1 mg / kg) once a day. Figure 8 The experimental results showed that the survival time of septic mice was significantly improved after treatment with GCA@NHS / GelMA-MNPs or GCA ( Figure 8 Middle B), liver and lung damage was alleviated ( Figure 8There was no significant difference in the therapeutic effects between GCA@NHS / GelMA-MNPs and GCA, but GCA@NHS / GelMA-MNPs could significantly reduce the number of administrations and provide a more convenient administration method, thereby reducing the risk of infection.
[0063] Example 7: GCA@NHS / GelMA-MNPs improve the phagocytic function of macrophages in septic mice
[0064] This example measured the effect of the microneedles prepared in Example 1 on improving the phagocytic function of macrophages in septic mice. A comparative sample, PBS@NHS / GelMA-MNPs, was prepared using the same method as the GCA@NHS / GelMA-MNPs in Example 1, except that an equal mass of PBS was used in place of GCA.
[0065] In this example, septic mice were treated with GCA@NHS / GelMA-MNPs and PBS@NHS / GelMA-MNPs for 3 days. Bone marrow-derived macrophages were then collected and co-cultured with fluorescent microspheres that could be phagocytosed by macrophages for 1 hour. The number of microspheres phagocytosed by macrophages in the two groups of mice was then measured using a fluorescence microscope. Figure 9 The results showed that macrophages in the GCA@NHS / GelMA-MNPs group were able to phagocytose more fluorescent microspheres than those in the PBS@NHS / GelMA-MNPs group. This experimental result indicates that GCA@NHS / GelMA-MNPs improve the phagocytic function of macrophages in septic mice.
[0066] In summary:
[0067] 1. The present invention provides a hydrogel sustained-release microneedle loaded with GCA protein, which can slowly release GCA protein through the skin and has a significant therapeutic effect on septic mice. Those skilled in the art know that the maintenance of the higher-order structure of the active protein is crucial for it to exert its pharmacological effects. At the same time, the higher-order structure of the active protein is very sensitive to changes in external physical and chemical properties. When the active protein is loaded on the hydrogel sustained-release microneedle, the presence of organic solvents and the cross-linking reaction between organic substances make the maintenance of the higher-order structure of the active protein extremely uncertain. This is the biggest challenge when using active proteins to prepare hydrogel sustained-release microneedles, and often leads to failure. The hydrogel sustained-release microneedle loaded with GCA protein of the present invention can not only slowly release the GCA protein cross-linked therein, but also the released GCA protein still has excellent pharmacological effects, and there is no significant difference in the therapeutic effect with the injection of GCA protein.
[0068] 2. Compared with intravenous injection, the GCA protein-loaded hydrogel sustained-release microneedles provided by the present invention can reduce the pain and discomfort of use, and are more convenient to store and carry, thereby improving patient compliance.
[0069] The purpose of the above embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.
Claims
1. A hydrogel sustained-release microneedle loaded with particulate calcineurin, characterized in that: The hydrogel sustained-release microneedle comprises: Base substrate: The base substrate has a microneedle side and an opposing back side; Solid microneedle: The solid microneedle extends from the microneedle side of the base substrate and includes a hydrogel polymer and granular calcium protein, wherein: the hydrogel polymer is obtained by cross-linking polymerization of low-temperature sensitive methacrylated gelatin and acrylated polyethylene glycol NHS ester with a photoinitiator, and the granular calcium protein is dispersed in the hydrogel polymer.
2. The hydrogel sustained-release microneedle according to claim 1, characterized in that: Calculated on the basis of the low-temperature sensitive methacrylated gelatin, 20 μg to 25 μg of acryloyl polyethylene glycol NHS ester is used for every 1.5 g of the low-temperature sensitive methacrylated gelatin.
3. The hydrogel sustained-release microneedle according to claim 1, characterized in that: Based on the low-temperature sensitive methacryloyl gelatin, 1 mg to 1.2 mg of granular calcium protein is used for every 1.5 g of low-temperature sensitive methacryloyl gelatin.
4. The hydrogel sustained-release microneedle according to claim 1, characterized in that: The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphate.
5. The hydrogel sustained-release microneedle according to claim 4, characterized in that: Calculated on the basis of the low-temperature sensitive methacryloyl gelatin, 0.03 g to 0.04 g of lithium phenyl-2,4,6-trimethylbenzoyl phosphate is used for every 1.5 g of the low-temperature sensitive methacryloyl gelatin.
6. The hydrogel sustained-release microneedle according to claim 1, characterized in that: The cross-linking polymerization conditions are: irradiation curing at 405nm to 410nm for 30s to 35s.
7. The hydrogel sustained-release microneedle according to claim 1, characterized in that: The thickness of the base substrate is 600 to 700 microns, and the length of the solid microneedles is 550 to 650 microns.
8. A method for preparing the hydrogel sustained-release microneedle according to any one of claims 1 to 7, characterized in that: The steps include: preparing a buffer solution containing a photoinitiator; dissolving the low-temperature sensitive methacrylated gelatin with a portion of the buffer solution to obtain a first mixed solution; adding granular calcein and acryloyl-polyethylene glycol NHS ester to the remaining buffer solution to obtain a second mixed solution; The first mixed solution and the second mixed solution are mixed, added into a microneedle mold, concentrated, cross-linked and solidified, dried, and demoulded to obtain the microneedle mold.
9. The preparation method according to claim 8, characterized in that: The buffer solution is phosphate buffer.
10. Use of the hydrogel sustained-release microneedle according to any one of claims 1 to 7 in the preparation of a drug or device for treating sepsis.