Anti-inflammatory plasma matrix as well as preparation method and application thereof
By mixing a plasma matrix with aspirin-chitosan nanoparticles to form an anti-inflammatory plasma matrix, the problem of insufficient anti-inflammatory capacity in existing technologies is solved, achieving better treatment results for periodontitis and peri-implantitis.
Patent Information
- Application Number
- CN202410604287.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-15
- Publication Date
- 2025-12-26
AI Technical Summary
Existing plasma matrix has insufficient anti-inflammatory capacity in the treatment of periodontitis and peri-implantitis, making it difficult to effectively control infection and promote tissue regeneration.
By mixing aspirin with chitosan nanoparticles to form an anti-inflammatory plasma matrix, the sustained-release and antibacterial effects of chitosan nanoparticles are combined with the anti-inflammatory and bone regeneration capabilities of aspirin to improve treatment efficacy.
It prolongs the treatment time of aspirin, enhances its anti-inflammatory and antibacterial effects, promotes the regeneration of soft and hard tissues, and provides better clinical treatment results.
Smart Images
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of blood products, in particular to an anti-inflammatory plasma matrix and a preparation method and application thereof. BACKGROUND
[0002] Periodontitis is a chronic inflammatory disease with bacteria as the main pathogenic factor. Recent theories attribute the pathogenesis of periodontitis to oral microbial dysbiosis, in which Porphyromonas gingivalis plays a key role by disrupting host immune homeostasis. Although local microbial community dysbiosis triggers local inflammation, overactivation of the host immune response directly activates osteoclast activity and alveolar bone loss. The cytokine network involved in periodontitis and its important and pleiotropic role in specific immune cell recruitment, pathogen control and induction or inhibition of osteoclast activity.
[0003] Plasma matrix is derived from blood and contains a variety of cytokines, including a large number of cytokines inside platelets, which can be slowly released into the surrounding environment. Some researchers have studied the anti-inflammatory effects of plasma matrix on immune response-related cells, especially macrophages and dendritic cells. The pro-inflammatory M1 phenotype of macrophages in bacterial suspension and the activation of dendritic cells around muscle defects. In addition, in vitro experiments also showed similar results. Plasma matrix to some extent eliminated the inflammatory response caused by lipopolysaccharide. The study found that the activation factor TLR4 of inflammatory stimulation and the key factor p-p65 of the classical inflammation-related NF-κB signaling pathway can be inhibited by plasma matrix. The results show that plasma matrix has potential anti-inflammatory effects in the process of regeneration and repair. However, the anti-inflammatory components contained in the plasma matrix itself are only derived from itself, and the content is low.
[0004] Therefore, it is necessary to seek a plasma matrix component with higher anti-inflammatory capacity for the control of periodontal and peri-implant infection, inflammation, and the promotion of soft and hard tissue regeneration to achieve good treatment effect. SUMMARY
[0005] In order to solve at least one of the above technical problems, the present application provides an anti-inflammatory plasma matrix and a preparation method and application thereof.
[0006] As one aspect, the present application provides an anti-inflammatory plasma matrix, comprising: a plasma matrix and aspirin chitosan nanoparticles.
[0007] As another aspect, the present application provides a preparation method of the anti-inflammatory plasma matrix as described above, comprising: pressing a mixture of the plasma matrix and aspirin chitosan nanoparticles to obtain the anti-inflammatory plasma matrix.
[0008] As a further aspect, the present application provides the use of the anti-inflammatory plasma matrix or the anti-inflammatory plasma matrix prepared by the method for preparing the anti-inflammatory plasma matrix in the preparation of a drug for periodontitis or peri-implantitis.
[0009] The technical solution of the embodiment of the present application has the following advantages:
[0010] In the anti-inflammatory plasma matrix of the embodiment of the present application, the chitosan nanoparticles can provide a slow and long-lasting drug release effect, prolong the action time of aspirin in the treatment area, and the chitosan itself has good antibacterial effect, further improving the infection control of periodontitis; aspirin itself has anti-inflammatory and bone regeneration regulation ability, combined with the advantages of chitosan nanoparticles, can produce a synergistic effect to improve the treatment effect. In addition, the plasma matrix is from the patient's own body, has good anti-inflammatory, antibacterial and regeneration effects, and as a carrier of nanoparticles, can play a role in slow release and keeping the nanoparticles in the local periodontitis or peri-implantitis.
[0011] In the anti-inflammatory plasma matrix of the embodiment of the present application, the white blood cells contained in the plasma matrix itself can play an antibacterial role, the aspirin chitosan nanoparticles can release aspirin, play a role in regulating bone regeneration cycle in the inflammatory state, and the chitosan shell also has certain antibacterial and anti-inflammatory effects. In addition, the anti-inflammatory plasma matrix can be used alone or mixed with other regeneration materials in the local periodontitis or peri-implantitis, which can assist in antibacterial and inflammation control, promote soft and hard tissue regeneration, and obtain better clinical effect. BRIEF DESCRIPTION OF DRAWINGS
[0012] The accompanying drawings, which are incorporated into and form a part of the specification, illustrate an embodiment consistent with the present application and, together with the description, serve to explain the principles of the application.
[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.
[0014] Figure 1 It is a transmission electron microscope image of the aspirin chitosan nanoparticles of the embodiment 1 of the present application.
[0015] Figure 2 It is a comparison chart of the change of cell inflammatory factors after being treated by the anti-inflammatory plasma matrix of the embodiment 1 of the present application and the solid plasma matrix of the comparative example 1, respectively. DETAILED DESCRIPTION
[0016] In order to enable a more clear understanding of the above-mentioned objects, features and advantages of the present application, the following will further describe the schemes of the present application. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.
[0017] In the following description, a large number of specific details are set forth in order to facilitate a thorough understanding of the present application, but the present application can also be implemented in other manners different from those described herein; obviously, the embodiments described in the specification are only a part of the embodiments of the present application, and not all the embodiments.
[0018] According to an embodiment of the present application, an anti-inflammatory plasma matrix is provided, comprising: a plasma matrix and aspirin chitosan nanoparticles.
[0019] In some embodiments of the present application, the mass-volume ratio of aspirin chitosan nanoparticles to plasma matrix is 50-150 mg / 10 ml; for example, any one ratio selected from 58 mg / 10 ml, 69 mg / 10 ml, 78 mg / 10 ml, 89 mg / 10 ml, 109 mg / 10 ml, 118 mg / 10 ml, 136 mg / 10 ml, 148 mg / 10 ml, etc.
[0020] In some embodiments of the present application, the plasma matrix contains fibrin three-dimensional structure, autologous living cells and active proteins.
[0021] In some embodiments of the present application, the preparation method of aspirin chitosan nanoparticles comprises: mixing aspirin solution and chitosan solution to react, using a terminating agent to terminate the reaction, and obtaining aspirin chitosan nanoparticles.
[0022] In some embodiments of the present application, the mixing method of aspirin chitosan nanoparticles and plasma matrix includes but is not limited to spraying on the inner surface of a blood collection tube, and then soaking, stirring, flicking, shaking, etc. to mix uniformly after obtaining the plasma matrix.
[0023] In some embodiments of the present application, the mixing method of aspirin chitosan nanoparticles and plasma matrix includes: one method is to put aspirin chitosan nanoparticles into a blood collection tube, and then mix them with plasma matrix (solid or liquid plasma matrix) by centrifugation; another method is to directly resuspend aspirin chitosan nanoparticles with liquid plasma matrix, and then mix them uniformly by stirring, flicking, etc.
[0024] In some embodiments of the present application, the preparation method of aspirin chitosan nanoparticles comprises: adding aspirin solution dropwise into chitosan solution to obtain a mixed solution, reacting, adding aqueous trisodium phosphate solution to terminate the reaction, and preparing aspirin chitosan nanoparticles.
[0025] In some embodiments of the application, in the preparation method of the aspirin-chitosan nanoparticles, the mass ratio of aspirin in the aspirin solution to chitosan in the chitosan solution is (0.025-0.875):1, for example, any one of the mass ratios selected from 0.078:1, 0.098:1, 0.183:1, 0.239:1, 0.386:1, 0.468:1, 0.536:1, 0.676:1, 0.789:1, 0.869:1, etc.
[0026] In some embodiments of the application, in the preparation method of the aspirin-chitosan nanoparticles, the aspirin solution is an ethanol solution of aspirin; and / or, the mass concentration of chitosan in the chitosan solution is 0.1-0.5%, for example, any one of the concentration values selected from 0.16%, 0.21%, 0.28%, 0.31%, 0.36%, 0.42%, 0.48%, etc.; and / or, the mass concentration of the aqueous solution of trisodium phosphate is 1.5-8%, for example, 1.9%, 2.3%, 2.9%, 3.7%, 4.3%, 5.8%, 6.9%, 7.8%, etc.
[0027] In some embodiments of the application, in the preparation method of the aspirin-chitosan nanoparticles, the chitosan solution is an aqueous acetic acid solution of chitosan.
[0028] In some embodiments of the application, in the preparation method of the aspirin-chitosan nanoparticles, the volume percentage of acetic acid in the aqueous acetic acid solution is 1-2%, for example, 1.1%, 1.3%, 1.4%, 1.6%, 1.8%, 1.9%, etc.
[0029] In some embodiments of the application, in the preparation method of the aspirin-chitosan nanoparticles, further comprising: storing the aspirin-chitosan nanoparticles at -80℃ to -20℃, for example, any one of the temperature values selected from -78℃, -58℃, -46℃, -36℃, -26℃, etc.
[0030] In some embodiments of the application, the preparation method of the aspirin-chitosan nanoparticles comprises the following steps:
[0031] S1, dissolving an appropriate amount of chitosan in an aqueous acetic acid solution to obtain a chitosan solution; wherein the volume ratio of the aqueous acetic acid solution (volume of acetic acid / volume of the aqueous acetic acid solution) is 1-2%, and the mass concentration of the chitosan solution (mass of chitosan / volume of the chitosan solution) is 0.1-0.5%.
[0032] S2, in another container, dissolving an appropriate amount of aspirin in anhydrous ethanol to form a uniform solution.
[0033] S3, different volumes of aspirin ethanol solution are slowly added dropwise into the chitosan solution, and uniform stirring is maintained with the help of a magnetic stirrer; wherein the volume percentage of the aspirin ethanol solution added into the reaction solution (aspirin solution + chitosan solution) (volume of aspirin solution / volume of reaction solution) is 0.1-0.5%.
[0034] S4, during magnetic stirring, the reaction is terminated by adding dropwise a trisodium phosphate (TPP) aqueous solution, and after stirring for 10 minutes, the final mixture is centrifuged, the supernatant is removed, and a nanoparticle precipitate is obtained.
[0035] S5, finally, the obtained aspirin-chitosan complex is repeatedly washed with distilled water, and then freeze-dried to obtain aspirin-chitosan nanoparticles; wherein the aspirin-chitosan nanoparticles after freeze-drying are stored at -80℃ to -20℃.
[0036] In some embodiments of the present application, the use form of the aspirin-chitosan nanoparticles includes: one is to use directly in the form of particles; and the other is to prepare into a solution form.
[0037] In some embodiments of the present application, the plasma matrix is prepared by centrifuging blood. Wherein the plasma matrix includes at least one of a solid plasma matrix or a liquid plasma matrix.
[0038] In some embodiments of the present application, the preparation method of the plasma matrix includes the following steps: centrifuging blood to obtain a solid plasma matrix or a liquid plasma matrix. Specifically, the solid plasma matrix obtained after centrifugation contains a plasma matrix clot, which needs to be taken out using tweezers or the like, and after removing the red blood cell layer, the plasma matrix is prepared by using a plasma matrix film pressing kit for pressing. The liquid plasma matrix obtained after centrifugation is prepared by using a pipette, a syringe or the like to suck the upper layer of light yellow plasma matrix and the middle layer. And / or, the centrifugal force of the blood centrifugation is 300-1000g, for example, the centrifugal force is selected from any one of 300g, 460g, 580g, 690g, 780g, 890g, 980g, 1000g, and the like, and is preferably 700g. And / or, the centrifugation time is 2-10min, for example, the centrifugation time is selected from any one of 3min, 4min, 6min, 7min, 8min, 9min, and the like, and is preferably 8min.
[0039] According to the embodiments of the present application, a preparation method of the anti-inflammatory plasma matrix as described above is provided, which includes: pressing the mixture of the plasma matrix and the aspirin-chitosan nanoparticles to obtain the anti-inflammatory plasma matrix.
[0040] According to an embodiment of the present application, there is provided an anti-inflammatory plasma matrix as described above or an anti-inflammatory plasma matrix prepared by the method as described above for use in the preparation of a medicament for periodontitis or peri-implantitis.
[0041] To further illustrate the effects of the present application, specific examples are provided below.
[0042] Example 1
[0043] A method for preparing an anti-inflammatory plasma matrix, comprising the following steps:
[0044] (1) Preparation of a chitosan solution
[0045] 0.5 g of chitosan was dissolved in 100 mL of 1% acetic acid aqueous solution to obtain a 0.5% chitosan solution.
[0046] (2) Preparation of an aspirin solution
[0047] In another beaker, 0.5 g of aspirin was dissolved in 100 mL of absolute ethanol to form a uniform solution.
[0048] (3) Preparation of aspirin-chitosan nanoparticles
[0049] 10 mL of the aspirin ethanol solution was slowly added dropwise to 50 mL of the chitosan solution, and uniform stirring was maintained with the help of a magnetic stirrer. During the magnetic stirring, the reaction was terminated by dropwise addition of 2% aqueous trisodium phosphate (TPP), and after stirring for 10 minutes, the final mixture was centrifuged to remove the supernatant, obtaining a nanoparticle precipitate. Finally, the aspirin-chitosan complex obtained was repeatedly washed with distilled water, and then freeze-dried to obtain aspirin-chitosan nanoparticles.
[0050] (4) Preparation of an anti-inflammatory plasma matrix
[0051] 0.5 g of aspirin-chitosan nanoparticles were added to a glass plasma matrix blood collection tube by spraying, and then 10 mL of a whole blood sample was added to obtain a mixed solution, which was quickly placed in a horizontal centrifuge and centrifuged at a centrifugal force of 700 x g for 8 min. The upper solid plasma matrix clot was bluntly separated from the lower red blood cell layer with scissors, and then pressed into a film shape with a special film pressing device box (Pleasure Shield), and placed in the surgical area in the clinic. An anti-inflammatory plasma matrix for the auxiliary treatment of periodontitis obtained has a lighter postoperative pain response.
[0052] The transmission electron microscope image of the aspirin-chitosan nanoparticles prepared in Example 1 is shown in Figure 1 The particle size of the aspirin-chitosan nanoparticles prepared in Example 1 is listed in Table 1.
[0053] Table 1 Particle size of aspirin-chitosan nanoparticles prepared in Example 1
[0054] Aspirin-chitosan nanoparticles Particle size 50 mg aspirin (100 mL 0.5 w / v chitosan solution) 91 ± 12.3 nm
[0055] Example 2
[0056] A method for preparing an anti-inflammatory plasma matrix, comprising the steps of:
[0057] (1) Preparation of chitosan solution
[0058] 0.2 g of chitosan was dissolved in 100 mL of 1% acetic acid aqueous solution to obtain a 0.2% chitosan solution.
[0059] (2) Preparation of aspirin solution
[0060] In another beaker, 0.4 g of aspirin was dissolved in 100 mL of absolute ethanol to form a uniform solution.
[0061] (3) Preparation of aspirin-chitosan nanoparticles
[0062] 5 mL of aspirin ethanol solution was slowly added dropwise to 20 mL of chitosan solution, and uniform stirring was maintained with the help of a magnetic stirrer. During the magnetic stirring, the reaction was terminated by adding dropwise a water solution of trisodium phosphate (TPP), and after stirring for 10 minutes, the final mixture was centrifuged, the supernatant was removed, and a nanoparticle precipitate was obtained. Finally, the aspirin-chitosan complex obtained was repeatedly washed with distilled water, and then freeze-dried to obtain aspirin-chitosan nanoparticles.
[0063] (4) Preparation of anti-inflammatory plasma matrix
[0064] 10 mL of whole blood sample was added to a plastic plasma matrix blood collection tube (Plasmacol), and the mixed solution was quickly placed in a horizontal centrifuge and centrifuged at a centrifugal force of 500 x g for 6 min to obtain an upper plasma matrix liquid. The upper yellow liquid was aspirated with a syringe and mixed uniformly with 0.1 g of freeze-dried aspirin-chitosan nanoparticles, and injected into the surgical area. An anti-inflammatory plasma matrix for the auxiliary treatment of periodontitis obtained has a lighter postoperative reaction, and postoperative swelling is controlled.
[0065] Example 3
[0066] A method for preparing an anti-inflammatory plasma matrix, comprising the steps of:
[0067] (1) Preparation of chitosan solution
[0068] 0.1 g of chitosan was dissolved in 100 mL of 1% acetic acid aqueous solution to obtain a 0.1% chitosan solution.
[0069] (2) Preparation of aspirin solution
[0070] In another beaker, 0.5 g of aspirin was dissolved in 100 mL of absolute ethanol to form a uniform solution.
[0071] (3) Preparation of aspirin-chitosan nanoparticles
[0072] The 5 mL aspirin ethanol solution was slowly added dropwise into the 30 mL chitosan solution, and uniform stirring was maintained with the help of a magnetic stirrer. During the magnetic stirring, the reaction was terminated by adding dropwise an aqueous solution of trisodium phosphate (TPP), and after stirring for 20 minutes, the final mixture was centrifuged, the supernatant was removed, and the nanoparticle precipitate was obtained. Finally, the aspirin-chitosan complex obtained was repeatedly washed with distilled water, and then freeze-dried to obtain aspirin-chitosan nanoparticles.
[0073] (4) Preparation of anti-inflammatory plasma matrix
[0074] The 50 mg aspirin-chitosan nanoparticles were added to the blood collection tube of the PAXgene Blood Plasma Glass Matrix by spraying, and then 10 mL of whole blood sample was added to obtain a mixed solution. The mixed solution was quickly placed in a horizontal centrifuge and centrifuged at a centrifugal force of 500 x g for 8 min to obtain an upper solid plasma matrix. The obtained upper solid plasma matrix clot was bluntly separated from the lower red blood cell layer with scissors, and then the obtained anti-inflammatory solid plasma matrix was obtained.
[0075] Comparative Example 1
[0076] The preparation method of the solid plasma matrix in this comparative example 1 is the same as the preparation method of the anti-inflammatory plasma matrix for the auxiliary treatment of periodontitis in Example 1, except that the aspirin-chitosan nanoparticles are not sprayed into the tube wall.
[0077] Performance evaluation
[0078] The solid plasma matrix of Comparative Example 1 and the anti-inflammatory solid plasma matrix of Example 1 were respectively placed in a 6-well plate, and 5 mL of fresh DMEM medium was added to each. The plates were placed in a cell culture incubator for 3 days. The medium containing the leachate was used to prepare a cell culture medium containing 10% fetal bovine serum. THP-1 cells were cultured and inoculated in a 12-well plate, and the prepared medium was added for culture for 7 days. Cell RNA was extracted by the Trizol method, cDNA was obtained by reverse transcription, and gene expression level detection was performed using a Bio-rad 96 PCR instrument. The test results were plotted in Figure 2 , wherein, Figure 2In this context, "control" refers to cell data cultured using only ordinary 10% fetal bovine serum cell culture medium; "plasma matrix" refers to data containing plasma matrix extract of "Comparative Example 1"; and "anti-inflammatory plasma matrix" refers to data containing plasma matrix extract of "Example 1".
[0079] according to Figure 2 The data on the relative expression levels of inflammatory factors after culturing cells using different plasma matrices shown lead to the following conclusions: The expression of the pro-inflammatory factor IL-1β showed a decrease in the plasma matrix group, and a further decrease in the anti-inflammatory plasma matrix group used as adjunctive therapy for periodontitis (Example 3). The expression of the anti-inflammatory factor IL-10 showed an increase in the plasma matrix group, and a further increase in the anti-inflammatory plasma matrix group used as adjunctive therapy for periodontitis (Example 3). In summary, the anti-inflammatory plasma matrix used as adjunctive therapy for periodontitis exhibits a better anti-inflammatory effect.
[0080] In summary, the anti-inflammatory plasma matrix provided in this embodiment of the invention contains aspirin-chitosan nanoparticles, which can utilize the anti-inflammatory and sustained-release effects of the plasma matrix itself, combined with the anti-inflammatory and bone regeneration effects of aspirin, and the antibacterial and anti-inflammatory effects of chitosan, thereby achieving better clinical results.
[0081] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.
[0082] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. An anti-inflammatory plasma matrix, characterized in that, include: Plasma matrix and aspirin chitosan nanoparticles.
2. The anti-inflammatory plasma matrix according to claim 1, characterized in that, The mass-to-volume ratio of the aspirin chitosan nanoparticles to the plasma matrix is 50–150 mg / 10 mL.
3. The anti-inflammatory plasma matrix according to claim 1 or 2, characterized in that, The preparation method of the aspirin-chitosan nanoparticles includes: mixing an aspirin solution and a chitosan solution to react, and terminating the reaction with a terminator to obtain the aspirin-chitosan nanoparticles.
4. The anti-inflammatory plasma matrix according to claim 3, characterized in that, In the method for preparing aspirin-chitosan nanoparticles, the mass ratio of aspirin in the aspirin solution to chitosan in the chitosan solution is (0.025–0.875):
1.
5. The anti-inflammatory plasma matrix according to claim 3 or 4, characterized in that, In the preparation method of the aspirin chitosan nanoparticles, The aspirin solution is an ethanol solution of aspirin; And / or, the chitosan solution contains 0.1% to 0.5% chitosan by mass. And / or, the mass concentration of the trisodium phosphate aqueous solution is 1.5% to 8%.
6. The anti-inflammatory plasma matrix according to any one of claims 3 to 5, characterized in that, In the method for preparing aspirin chitosan nanoparticles, the chitosan solution is an aqueous solution of chitosan in acetic acid.
7. The anti-inflammatory plasma matrix according to claim 6, characterized in that, In the preparation method of the aspirin chitosan nanoparticles, the volume percentage of acetic acid in the aqueous acetic acid solution is 1-2%.
8. The anti-inflammatory plasma matrix according to any one of claims 3 to 7, characterized in that, The method for preparing aspirin chitosan nanoparticles further includes storing the aspirin chitosan nanoparticles at -80℃ to -20℃.
9. A method for preparing an anti-inflammatory plasma matrix as described in any one of claims 1 to 8, characterized in that, include: The anti-inflammatory plasma matrix is obtained by pressing a mixture of plasma matrix and aspirin chitosan nanoparticles.
10. The use of an anti-inflammatory plasma matrix prepared by any one of claims 1 to 8 or by the method of preparing the anti-inflammatory plasma matrix according to claim 9 in the preparation of a drug for periodontitis and peri-implantitis.