RNA particle complex, method for producing the same, and its applications.

Abstract

This invention relates to an RNA particle complex, a method for producing the same, and its applications. The RNA particle complex comprises RNA and carbonate apatite particles, and its production method includes the following steps: S1, 300 μg of RNA, 440 mM NaHCO3, 9 mM NaH2PO4, and 58 mM CaCl2 are added to prepare 100 mL of an aqueous solution containing RNA and an inorganic ion mixture; S2, after stirring at 20°C for 3 minutes, the mixture is centrifuged at 4000 rpm for 3 minutes, and the resulting precipitate is the RNA particle complex. The invention also includes the application of the RNA particle complex to drugs or daily necessities for the treatment of dermatitis and the promotion of skin wound healing. When applied to skin drugs or daily necessities, this RNA particle complex can significantly improve the therapeutic effect on skin wound healing and effectively prevent scar formation caused by skin damage.

Description

[Technical Field]

[0001] This invention relates to the biomedical technology field, and more particularly to RNA particle complexes, methods for producing the same, and applications thereof. [Background technology]

[0002] RNA, or ribonucleic acid, is a carrier of genetic information found in biological cells and some viruses and viroids. It is formed by the linking of at least several dozen ribonucleotides by phosphodiester bonds and its name derives from the presence of ribose. RNA is universally present in animals, plants, microorganisms, and certain viruses and bacteriophages. RNA is closely related to protein biosynthesis. In RNA viruses and bacteriophages, RNA is the carrier of genetic information.

[0003] In the prior art, for example, the Chinese patent with publication number CN108697653B relates to wound treatment and therapy after injury and to promoting tissue regeneration. Specifically, it discloses a conjugate of miRNA-146a and nanocerium oxide for improving wound healing and, in some embodiments, preventing poor ventricular remodeling after myocardial infarction.

[0004] Although the aforementioned miRNA-146a has anti-inflammatory and wound healing-promoting effects, there are currently no products that use miRNA-146a as the main ingredient in skin medications or daily chemicals. The reasons for this are as follows: 1. miRNA is easily degraded. 2. The skin penetration of the active ingredient is low. 3. There are no effective RNA delivery vectors that can deliver large amounts of RNA to the skin. [Overview of the project]

[0005] The object of the present invention is to provide an RNA particle complex, a method for producing the same, and its applications. This RNA particle complex can significantly improve the therapeutic effect on skin wound healing and effectively prevent scar formation caused by skin trauma when used in skin drugs or daily chemicals.

[0006] The above objectives of the present invention are achieved by the following technical solution: RNA particle complexes contain RNA and carbonate apatite particles.

[0007] In a preferred embodiment, the mass percentage of the RNA is 0.005% to 1.5%, and the mass percentage of the carbonate apatite particles is 98.5% to 99.995%.

[0008] In a preferred embodiment, the mass percentage of the RNA is 1.5% to 5%, and the mass percentage of the carbonate apatite particles is 95% to 98.5%.

[0009] In a preferred embodiment, the mass percentage of the RNA is 5% to 15%, and the mass percentage of the carbonate apatite particles is 85% to 95%.

[0010] In a preferred embodiment, the RNA is any one or more combinations selected from the group consisting of microRNA (miRNA), siRNA, messenger RNA (mRNA), long non-coding RNA (lncRNA), piRNA, and snoRNA.

[0011] The present invention further provides a method for producing the above RNA particle complex. This method includes the following steps: S1: Prepare an aqueous solution containing RNA and an inorganic ion mixture. S2: The aqueous solution is stirred and uniformly mixed, then centrifuged, and the resulting precipitate is considered an RNA particle complex.

[0012] Selectively, the method for producing the above RNA particle complex includes the following steps: S1: Add 300 μg of RNA, 440 mM NaHCO3, 9 mM NaH2PO4, and 58 mM CaCl2 to prepare 100 mL of aqueous solution containing RNA and inorganic ion mixture. S2: After stirring at 20°C for 3 minutes, the mixture is centrifuged at 4000 rpm for 3 minutes, and the resulting precipitate is considered the RNA particle complex.

[0013] In one preferred embodiment, in step S1, the inorganic ion mixture is Ca2 + CO3 2- , PO4 3- na + Cl - Includes.

[0014] Selectively, the inorganic ion mixture contains carbonate apatite particles.

[0015] One embodiment of the present invention provides that the RNA particle complex or the RNA particle complex obtained by the above manufacturing method is used for the treatment of dermatitis.

[0016] One embodiment of the present invention provides that the RNA particle complex described above or the RNA particle complex obtained by the above manufacturing method is used to promote the healing of skin wounds.

[0017] One embodiment of the present invention provides that the RNA particle complex or the RNA particle complex obtained by the above manufacturing method is used in daily chemical products.

[0018] The present invention further provides a drug for treating dermatitis, comprising the above-mentioned RNA particle complex or an RNA particle complex obtained by the above-mentioned manufacturing method.

[0019] The present invention further provides a pharmaceutical product for promoting skin wound healing, comprising the above-mentioned RNA particle complex or an RNA particle complex obtained by the above-mentioned manufacturing method.

[0020] The present invention further provides a daily chemical product comprising the above RNA particle complex or the RNA particle complex obtained by the above production method.

[0021] The present invention further provides use of the above RNA particle complex or the RNA particle complex obtained by the above production method in the preparation of a medicament for treating dermatitis.

[0022] The present invention further provides use of the above RNA particle complex or the RNA particle complex obtained by the above production method in the preparation of a medicament for promoting skin wound healing.

[0023] The present invention further provides use of the above RNA particle complex or the RNA particle complex obtained by the above production method in the preparation of a daily chemical product.

[0024] The present invention further provides a method for treating dermatitis, comprising administering an effective amount of the above RNA particle complex or the RNA particle complex obtained by the above production method to a subject in need thereof.

[0025] The present invention further provides a method for promoting skin wound healing, comprising administering an effective amount of the above RNA particle complex to a subject in need thereof.

[0026] As a preferred embodiment of the present invention, when applied in skin medicaments or daily chemical products, the RNA particle complex can significantly improve the therapeutic effect on skin wound healing and effectively prevent scar formation caused by skin trauma. Herein, the skin trauma includes burns of the superficial skin layer and trauma of the deep skin layer. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] [Figure 1] Schematic overview of the RNA particle complex. [Figure 2] Shows the substance stability of RNA (50 µg of miRNA146a-5p) and the RNA particle complex (containing 50 µg of miRNA146a-5p) in mouse serum. [Figure 3]Overview of the creation of the allergic dermatitis model in Experiment Example 1. [Figure 4] Healing status of dorsal dermatitis in mice in Experimental Example 1. [Figure 5] The statistical table for evaluating skin lesion scores on Day 0 and Day 4 in Experiment Example 1 is shown. [Figure 6] The statistical table of changes in skin inflammation lesion scores in Experimental Example 1 is shown. [Figure 7] The HE staining analysis of mouse dermatitis pathology in Experimental Example 1 is shown. [Figure 8] This shows the healing status of full-thickness skin injury on the back of mice in Experimental Example 2. [Figure 9] The statistics for evaluating skin wound healing on Day 15 in Experimental Example 2 are shown. [Figure 10] The HE staining analysis of mouse dermatitis pathology in experimental example two is shown. [Figure 11] The immunohistochemical staining diagram of collagen-1 from experimental example two is shown. [Figure 12] The immunohistochemical staining image of FGF-2 in experimental example two is shown. [Modes for carrying out the invention]

[0028] The present invention will be illustrated below through specific examples. Persons of the art in this area should understand that the following examples are merely aids to understanding the present invention and should not be considered to limit its scope.

[0029] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as those commonly understood by those skilled in the art to which this invention pertains.

[0030] In this invention, "content" means calculated by weight unless otherwise specified. Technologists in the art will be able to understand the specific meaning of the above terms in this application depending on the specific circumstances.

[0031] miRNA146a: MicroRNA (miR)-146a regulates autoimmunity, inflammation, and keratinocyte proliferation, and is involved in the pathogenesis of psoriasis (Shen, H., et al., J Clin Lab Anal, 2022. 36(2): p. e24198.). In human primary keratinocytes, miR-146a suppresses the expression of many pro-inflammatory factors, including CCL5 and IL-8 (Meisgen, F., et al., J Invest Dermatol, 2014. 134(7): p. 1931-1940; Rebane, A., et al., J Allergy Clin Immunol, 2014. 134(4): p. 836-847 e11). miR-146a has been shown to act as an anti-inflammatory miRNA in mouse models of allergic dermatitis[3], irritant contact dermatitis[4], and psoriasis[5] (Rebane, A., et al., J Allergy Clin Immunol, 2014. 134(4): p. 836-847 e11; Urgard, E., et al., J Control Release, 2016. 235: p. 195-204; Srivastava, A., et al., J Allergy Clin Immunol, 2017. 139(2): p. 550-561). Simultaneously, miR-146a plays a crucial role in diabetic wound healing by regulating pro-inflammatory gene expression, and regulation of miR-146a levels through mesenchymal stem cell therapy can reduce wound inflammation and enhance wound closure (Xu, J., et al., Diabetes, 2012. 61(11): p. 2906-12). Engineered exosomes carrying MiR-146a target IRAK1 and promote diabetic wound healing (Li, Q., et al., Signal Transduct Target Ther, 2023. 8(1): p. 62).

[0032] Nanoparticle RNA delivery system: In 2015, inventors Wu, X., et al. first reported a novel nanoparticle system made of inorganic ions called supercarbonate apatite as an in vivo delivery system for intravenous administration of siRNA / miRNA (Wu, X., et al., PLoS One, 2015. 10(3): p. e0116022). Subsequently, this delivery system has been reported to be applicable to mouse models of several cancer therapies based on siRNA or miRNA (Takeyama, H., et al., Mol Cancer Ther, 2014. 13(4): p. 976-85; Takahashi, H., et al., Mol Cancer Ther, 2015. 14(7): p. 1705-16; Ogawa, H., et al., PLoS One, 2015. 10(5): p. e0127119; Hiraki, M., et al., Mol Ther Nucleic Acids, 2015. 4: p. e231; Inoue, A., et al., Mol Cancer Ther, 2018. 17(5): p. 977-987; Morimoto, Y., et al., Br J Cancer, 2020. 122(7): (pp. 1037-1049). In addition to cancer treatment, sCAs systemically deliver miRNAs to inflammatory lesions in the body and have been shown to treat inflammatory bowel disease (Fukata, T., et al., Mol Ther Nucleic Acids, 2018. 12: p. 658-671). Local delivery of sCAs containing plasmid DNA or siRNA to skin wounds can accelerate wound healing and reduce scar formation, demonstrating that this is an effective method for treating refractory abnormal scarring (Aoki, M., et al., Int J Mol Sci, 2019. 20(14); Aoki, M., et al., Mol Ther Nucleic Acids, 2020. 22: p. 50-61).

[0033] Skin tissue penetrating microparticle complex: Subsequently, through continuous improvement, Wu Xin has developed a microparticle complex for use in pharmaceuticals or daily chemicals. This complex aggregates bamboo sap extract, increases skin penetration, and has remarkable hair growth promoting effects (Application No.: CN202310728235.1, June 20, 2023), anti-inflammatory skin effects (Application No.: CN202310985360.0, August 7, 2023), and skin wound healing effects (Application No.: CN202310985076.3, August 7, 2023).

[0034] Embodiment 1: Referring to Figure 1, one embodiment of the RNA particle complex of the present invention includes RNA and carbonate apatite particles. The mass percentage of RNA is 0.005% to 1.5%, and the mass percentage of carbonate apatite particles is 98.5% to 99.995%. In this embodiment, the mass percentage of RNA is preferably 0.005%, and the mass percentage of carbonate apatite particles is preferably 99.995%. In this embodiment, RNA is any one or more types selected from the group consisting of microRNA (miRNA), siRNA, messenger RNA (mRNA), long non-coding RNA (lncRNA), piRNA, and snoRNA.

[0035] Embodiment 2: In one embodiment of the RNA particle complex of the present invention, RNA and carbonate apatite particles are included. The mass percentage of RNA is 1.5% to 5%, and the mass percentage of carbonate apatite particles is 95% to 98.5%. In this embodiment, RNA is any one or more types selected from the group consisting of microRNA (miRNA), siRNA, messenger RNA (mRNA), long non-coding RNA (lncRNA), piRNA, and snoRNA.

[0036] Embodiment 3: In one embodiment of the RNA particle composite of the present invention, the composite comprises RNA and carbonated apatite particles. The mass percentage of RNA is 5% to 15%, and the mass percentage of carbonated apatite particles is 85% to 95%. In this embodiment, the RNA is any one or a combination of two or more selected from the group consisting of microRNA (miRNA), siRNA, messenger RNA (mRNA), long non-coding RNA (lncRNA), piRNA and snoRNA.

[0037] Embodiment 4: The method for producing the RNA particle composite according to Embodiment 1 described above may comprise the following steps: S1: adding 300 μg of RNA (miRNA146a-5p), 440 mM of NaHCO3, 9 mM of NaH2PO4 and 58 mM of CaCl2 to prepare 100 mL of an aqueous solution containing a mixture of RNA and inorganic ions; S2: after stirring at 20°C for 3 minutes, centrifuging at 4000 rpm for 3 minutes, and collecting the resulting precipitate as the RNA particle composite. Herein, in step S1, the inorganic ion mixture comprises Ca2 + , CO3 2- , PO4 3- , Na + , Cl - . Referring to Figure 2, Figure 2 shows the substance stability of RNA (containing 50 μg of miRNA-146a-5p) and the RNA particle composite (containing 50 μg of miRNA-146a-5p) in mouse serum. When incubated in mouse serum at 37°C, RNA is degraded to about 20% of the original amount in only 10 minutes.

[0038] Embodiment 5: The RNA particle composite according to Embodiment 1 is applied to the treatment of dermatitis.

[0039] Embodiment 6: The RNA particle composite according to Embodiment 1 is applied to a medicament for promoting skin wound healing.

[0040] Embodiment 7: The RNA particle complex of Embodiment 1 is applied to everyday chemical products.

[0041] The present invention will be further explained in Experimental Examples 1 and 2 with reference to Figures 3 to 12:

[0042] Here, the HE staining method can be one that is commonly used in the field. For example, the procedure for an HE staining experiment is as follows: Section → Dry heat (for sectioning) → Deparaffinization and water displacement → Staining → Microscopic examination. HE staining experiment procedure: 1. Paraffin Sections: Fixed tissue samples are dehydrated in 75%, 85%, 95% I, 95% II, 100% I, and 100% II ethanol solutions for 60 minutes each, then cleared in xylene I and II for 45 minutes each, followed by immersion in paraffin I, II, and III for 50-60 minutes each, and then sectioned after paraffin embedding. 2. Deparaffinization and water displacement of paraffin sections: Dry heat the paraffin sections, then immerse them in xylene I for 10 minutes, xylene II for 10 minutes, anhydrous ethanol I for 3 minutes, anhydrous ethanol II for 3 minutes, 95% alcohol for 3 minutes, 80% alcohol for 3 minutes, and pure water for 2 minutes. 3. Hematoxylin staining: Immerse in hematoxylin staining solution for 3-5 minutes, then wash with tap water. Differentiate with 1% hydrochloric acid ethanol, then wash with tap water. Return to blue with blue dye, then wash with running water. 4. Eosin staining: Immerse the sections in eosin staining solution for 3-5 minutes, then wash with running water. 5. Dehydration and mounting: The sections are rapidly dehydrated by sequentially immersing them in 80%, 95%, 100% I, and 100% II ethanol solutions, and then mounted with a high-purity mounting medium. 6. Microscopy: Take images.

[0043] Explanation of staining results: The cell nucleus is stained blue, and the cytoplasm is stained red.

[0044] The immunohistochemical detection experiment described above can employ methods commonly used in the field. For example, an immunohistochemical experiment using paraffin sections can be employed, and the procedure is as follows: Paraffin-embedded sections → dry heat → deparaffinization and water replacement → antigen retrieval → removal of endogenous peroxidase → blocking → primary antibody incubation → secondary antibody incubation → DAB staining → counterstaining → dehydration and clearing → mounting → microscopy.

[0045] 1. Paraffin Sections: Fixed tissue samples are dehydrated in 75%, 85%, 95% I, 95% II, 100% I, and 100% II ethanol solutions for 60 minutes each. After clearing with xylene I and II for 45 minutes each, the samples are impregnated with paraffin I, II, and III for 50-60 minutes each. After paraffin embedding, sections are prepared.

[0046] 2. Deparaffinization and water displacement of paraffin sections: Dry heat the paraffin sections, then immerse them in xylene I for 10 minutes, xylene II for 10 minutes, anhydrous ethanol I for 3 minutes, anhydrous ethanol II for 3 minutes, 95% alcohol for 3 minutes, 80% alcohol for 3 minutes, and pure water for 2 minutes.

[0047] 3. Antigen retrieval of tissue sections (high pressure method): Place the tissue sections in a reaction vessel and add the antigen retrieval solution (citrate buffer). Heat in a pressure cooker and wait until the steam is automatically released. Then, let it stand for 2 minutes, remove from the heat source, and allow to cool naturally. Discard the antigen retrieval solution and wash the tissue sections with PBS.

[0048] 4. Removal of endogenous peroxidase: Transfer the sections to a humid chamber and add freshly prepared 3% hydrogen peroxide solution to inactivate the endogenous peroxidase. After incubation at room temperature for 10 minutes, wash thoroughly with PBS.

[0049] 5. Non-specific blocking: Wash the slide glass three times by immersing it in PBS for 5 minutes each time. Absorb the PBS around the tissue with filter paper, add 5% BSA drops onto the slide glass, and block at 37°C for 30 minutes.

[0050] 6. Primary antibody reaction: Absorb the blocking fluid surrounding the tissue with filter paper, and do not wash. Add an appropriate amount of diluted primary antibody (e.g., VEGF (1 / 100), IGF-1 (1 / 100)) to each slide glass and incubate overnight at 4°C in a humid chamber.

[0051] 7. Secondary antibody reaction: Remove the humidified chamber that has been incubated overnight at 4°C and allow it to stand at room temperature for 45 minutes. Wash the slide glass three times with PBS for 5 minutes each time. Add diluted (1:100) horseradish peroxidase-labeled goat anti-rabbit IgG (H+L), incubate at 37°C for 30 minutes, and then wash thoroughly with PBS.

[0052] 8. Color development and counterstaining: Allow to develop color in DAB solution for 3-5 minutes, and check the degree of staining under a microscope. Wash with PBS or tap water for 1 minute, then counterstain with hematoxylin for 3 minutes. Differentiate with hydrochloric acid alcohol, return to blue with a blue dye solution, and wash with tap water.

[0053] 9. Dehydration, clarification, and mounting: The sections are rapidly dehydrated by sequentially immersing them in 80%, 95%, 100% I, and 100% II ethanol solutions. After clarification with xylene I and II, they are mounted with a neutral resin mounting medium and photographed under a microscope.

[0054] Explanation of staining results: Hematoxylin staining stains cell nuclei blue, while positive reactions detected by DAB staining stain yellowish-brown.

[0055] Experimental Example 1: 1. Introduction to the creation of allergic dermatitis models: As shown in Figure 3, the dorsal hair of mice was removed, and a model was induced using DNCB (2,4-dinitrochlorobenzene). On day 1, initial sensitization was performed by administering 150 μL of 2% DNCB solution to the back and 10 μL behind the ear. On days 5, 7, 9, 11, 13, 16, 18, 20, 22, and 24, stimulation was applied by administering 150 μL of 0.5% DNCB solution to the back and 10 μL behind the ear. On day 25, the model development status was evaluated by pathological section examination, skin lesion scoring, and scratching behavior test, and mice with a total score of 5 or higher were considered to have successfully developed the model.

[0056] Pathological examination of the tissue sections revealed that the skin tissue structure of the control group was normal under a light microscope, with no significant pathological changes observed. In contrast, the skin tissue of the model group showed keratinization in the stratum corneum, clear thickening of the epidermis, and infiltration of inflammatory cells in the dermis.

[0057] 2. Research experiments on the therapeutic effect of RNA particle complexes on mouse DNCB-induced dermatitis: Experimental animal: C57BL / 6 mouse, male, 6 weeks old; Experimental environment: Temperature 20-26°C, humidity 40-70%; Experimental grouping: The experiment was divided into three groups, with four animals in each group. 1) RNA group (2 sprays / animal / day): RNA aqueous solution containing the RNA content used in step S1 when preparing the RNA particle complex in Embodiment 4; 2) RNA particle complex group (2 sprays / animal / day): The precipitate obtained in step S2 of Embodiment 4 above was dissolved in 50 mL of distilled water and used as a spray; 3) Apatite aqueous solution group (2 sprays / animal / day): Apatite, which is carbonate apatite, is dissolved in water with 440 mM NaHCO3, 9 mM NaH2PO4, and 58 mM CaCl2 to make a total volume of 100 mL solution. After stirring at 20°C for 3 minutes, the solution is centrifuged at 4000 rpm for 3 minutes, and the resulting precipitate is dissolved in 50 mL of distilled water; Animal Model Creation: After acclimatizing all animals in animal pens, the dorsal hair of the mice was removed, and the model was induced using DNCB. On day 1, initial sensitization was performed by administering 150 μL of 2% DNCB solution to the back and 10 μL behind the ear. On days 5, 7, 9, 11, 13, 16, 18, 20, 22, and 24, stimulation was applied by administering 150 μL of 0.5% DNCB solution to the back and 10 μL behind the ear. On day 25, the model development status was evaluated by skin lesion scoring and scratching behavior tests, and mice with a total score of 5 or higher were considered to have successfully developed the model. Treatment process: Drug treatment corresponding to each group of animals was initiated on Day 0. According to the group division, two sprays of the drug were applied to the skin lesion and continued until euthanasia. The condition of the dorsal skin was observed by photography at two-day intervals on the day drug administration began (Day 0) and on days 2, 4, 6, and 8. After the observation period, the mice in each group were euthanized, tissue samples were taken from the injured areas, fixed with fixative, and then subjected to pathological HE staining.

[0058] The healing status of mouse dorsal dermatitis is shown in Figure 4: Figure 5 shows a statistical table of skin lesion scores for Day 0 and Day 4. The skin lesion score consisted of four parts: edema, hemorrhage, scaling, and epidermal shedding, and each part was evaluated on a scale from 0 (asymptomatic) to 3 (severe) according to its severity.

[0059] Figure 6 shows a line statistical graph illustrating the changes in the dermatitis lesion score of mice. The total inflammation score on Day 4 is as shown in Figure 6, and a significant anti-inflammatory effect on the skin was observed in the RNA particle complex treatment group compared to the RNA group and the apatite group. Statistical values ​​are shown as mean ± standard error. One-way ANOVA and Tukey's multiple comparison test were used for statistical evaluation, and a statistically significant difference was determined when P < 0.05.

[0060] Sampling: On day 8, skin tissue from the lower body of mice was collected under anesthesia, and after sectioning, pathological HE staining analysis was performed (see Figure 7). Observation under a light microscope revealed hyperkeratosis and hypokeratosis in the stratum corneum of the skin tissue in the RNA group and the apatite drug-treated group, and infiltration of numerous inflammatory cells in the dermis. On the other hand, after RNA particle complex drug treatment, inflammatory cell infiltration in the dermis was clearly reduced, keratinization of the stratum corneum improved, and the epidermis became thinner.

[0061] Experimental Example 2: Experimental environment: Temperature 20-26°C, humidity 40-70%; Experimental grouping: The experiment was divided into three groups, with four animals in each group. 1) RNA group (2 sprays / animal / day): An RNA aqueous solution containing the RNA content used in step S1 when preparing the RNA particle complex in Embodiment IV. 2) RNA particle complex group (2 sprays / animal / day): The precipitate obtained in step S2 of Embodiment IV above was dissolved in 50 mL of distilled water and used as a spray. 3) Apatite aqueous solution group (2 sprays / animal / day): Apatite, which is carbonate apatite, is dissolved in water with 440 mM NaHCO3, 9 mM NaH2PO4, and 58 mM CaCl2 to make a total volume of 100 mL solution. The solution is stirred at 20°C for 3 minutes, then centrifuged at 4000 rpm for 3 minutes, and the resulting precipitate is dissolved in 50 mL of distilled water. Animal model creation: On Day 0, animals were anesthetized and hair was removed from their backs. In the remaining groups, excluding the normal group, a full-thickness skin injury model was created, with a single 10 mm x 10 mm square injury on the lower back of each mouse. According to the group assignment, two sprays of the drug were applied to each wound and this was continued until sacrifice.

[0062] Photographic observation of wounded animals began on the day the model was created, and the wound condition was photographed and observed once every two days. Specifically, the wound condition was observed once on the day the model was created, and once each on the 2nd, 4th, 6th, 8th, 10th, 12th, 14th, 15th, 16th, 17th, and 18th days after the model was created. After the observation was completed, mice from each group were sacrificed, tissue from the injured site was collected, fixed with fixative, and then pathological HE staining and immunohistochemical detection for collagen-1 and FGF-2 expression were performed.

[0063] Experimental results and analysis: Figure 8 shows the healing status of full-thickness skin injury on the back of a mouse.

[0064] Figure 9 shows the evaluation of skin wound healing on Day 15. Compared to the RNA group and the apatite group, the RNA particle complex treatment group showed a significant improvement in skin wound healing. Statistical values ​​are shown as mean ± standard error. One-way ANOVA and Tukey's multiple comparison test were used for statistical evaluation, and a statistically significant difference was determined if P < 0.05.

[0065] Figure 10 shows the HE staining analysis of mouse skin pathology. In the RNA group and the apatite treatment group, keratinization was observed in the stratum corneum of the skin tissue of the model group, the epidermis was clearly thickened, and infiltration of inflammatory cells was observed in the dermis. After RNA particle complex treatment, the degree of keratinization in the stratum corneum of the skin was clearly improved, the epidermis was clearly thinned, and inflammatory cell infiltration was also reduced.

[0066] Figure 11 shows the expression of Collagen-1 as detected by immunohistochemical analysis. Compared to the RNA group and the apatite group, Collagen-1 expression was observed in dermal cells in the RNA particle complex group.

[0067] Figure 12 shows the expression of FGF-2 detected by immunohistochemical analysis. Compared to the RNA group and the apatite group, FGF-2 expression was observed in dermal cells in the RNA particle complex group.

[0068] Collagen is the most important component of the extracellular matrix, playing a role in connective tissue in animal cells as a polymer that makes up various extracellular stromas, and is also the most important structural protein in animal structural tissues. Skin is elastic mainly because the collagen in collagen fibers in the dermis layer forms the skin's skeleton. As we age and due to busy work and fatigue, the secretion of skin collagen decreases, causing the once soft skin to sag, dry out, and develop problems such as wrinkles and loss of luster. Therefore, to maintain youthful skin and vitality, it is necessary to increase the collagen content in the skin.

[0069] FGF2 plays an active pathophysiological role in tissue remodeling, bone health, and regeneration, contributing to, for example, the repair of nerve damage, the healing of skin wounds, joint protection, and the control of hypertension. Currently, clinical trials on FGF2 recombinant protein therapy are mainly focused on the repair of diseases such as oral diseases, periodontitis, tympanic membrane perforation, and age-related macular degeneration.

[0070] The above experimental results indicate the following: RNA particle complexes can enhance the therapeutic effect on skin wound healing and effectively prevent scar formation caused by skin trauma. Skin trauma here includes burns to the superficial layer of the skin and trauma to the deeper layers of the skin.

Claims

1. A type of RNA particle complex characterized by containing RNA and carbonate apatite particles.

2. The RNA particle complex according to claim 1 is characterized in that the mass percentage of the RNA is 0.005% to 1.5%, and the mass percentage of the carbonate apatite particles is 98.5% to 99.995%.

3. The RNA particle complex according to claim 1 is characterized in that the mass percentage of the RNA is 1.5% to 5%, and the mass percentage of the carbonate apatite particles is 95% to 98.5%.

4. The RNA particle complex according to claim 1 is characterized in that the mass percentage of the RNA is 5% to 15%, and the mass percentage of the carbonate apatite particles is 85% to 95%.

5. The RNA particle complex according to any one of claims 1 to 4 is characterized in that the RNA is one or a combination of several types selected from the group consisting of miRNA, siRNA, mRNA, lncRNA, piRNA, and snoRNA.

6. A method for producing an RNA particle complex according to any one of claims 1 to 5, the features comprising the following steps: S1. Prepare an aqueous solution containing RNA and an inorganic ion mixture; S2. The aqueous solution is stirred to ensure uniform mixing, then centrifuged, and the resulting precipitate is considered an RNA particle complex.

7. A method for producing an RNA particle complex according to any one of claims 1 to 5, comprising the following steps: S1, 300 μg RNA, 440 mM NaHCO3 3 , 9 mM NaH 2 PO 4 , and 58 mM CaCl 2 Add the following to prepare 100 mL of aqueous solution containing RNA and an inorganic ion mixture; After stirring at 20°C for 3 minutes, the mixture is centrifuged at 4000 rpm for 3 minutes, and the resulting precipitate is considered the RNA particle complex.

8. The method for producing an RNA particle complex according to claim 6 or 7, wherein in step S1, the inorganic ion mixture is Ca 2+ , CO 3 2+ , PO 4 3- , Na + , Cl - characterized in that it comprises. Optionally, the inorganic ion mixture comprises carbonate apatite particles.

9. Use of an RNA particle complex according to any one of claims 1 to 5, or an RNA particle complex produced by a manufacturing method according to any one of claims 6 to 8, for the treatment of dermatitis, promotion of skin wound healing, or as a daily necessities.

10. A daily necessities product or an agent for treating dermatitis or promoting the healing of skin wounds, comprising an RNA particle complex according to any one of claims 1 to 5, or an RNA particle complex produced by a manufacturing method according to any one of claims 6 to 8.

11. A method for treating dermatitis or promoting skin wound healing, comprising administering to a subject an effective amount of the RNA particle complex described in any one of claims 1 to 5, or the RNA particle complex produced by the manufacturing method described in any one of claims 6 to 8.

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