Preparation of HA-PEI-miR-146a nano composite material and anti-inflammatory application in periodontal treatment

Abstract

The invention belongs to the technical field of medicines, and particularly relates to preparation of an HA-PEI-miR-146a nano composite material and an anti-inflammatory application of the HA-PEI-miR-146a nano composite material in periodontal treatment. The invention designs a preparation method of a HA-PEI-miR-146a composite material, the HA-PEI-miR-146a composite material utilizes the characteristics of weakening potential, easy endocytosis, good stability and targeting property, low toxic and side effects, high biocompatibility, reduction of phagocytosis of an in-vivo immune system and the like of HA-PEI, and the HA-PEI-miR-146a composite material is combined with miR-146a with an anti-inflammatory effect, so that the HA-PEI-miR-146a composite material is prepared. And the cell permeability of the miR-146a is improved by virtue of the HA-PEI. Finally, the HA-PEI-miR-146a nano composite material disclosed by the invention has the advantages of easiness in transfection and good transfection effect, so that the miR-146a plays a role in inhibiting inflammation in periodontal clinical treatment.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to the preparation of a HA-PEI-miR-146a nanocomposite material and its anti-inflammatory application in periodontal treatment. Background Art

[0002] At present, the main treatment method for periodontal disease is to control periodontal inflammation through basic periodontal treatment, supplemented by laser treatment. However, this type of treatment method still cannot effectively control periodontal inflammation in special anatomical parts or special cases, and local medications are still needed for control when necessary. However, the current local medications also have limitations. Although they help control inflammation, they are not conducive to the growth of periodontal tissues. This is a difficult point in clinical treatment and a clinical problem that needs to be overcome. Therefore, it is necessary to seek an effective periodontal treatment method.

[0003] Lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg) is an important pathogenic factor of periodontal disease, which can induce host cells to secrete various inflammatory factors, leading to inflammatory response and damage to periodontal tissues. MicroRNAs (miRs) are a class of endogenous non-coding small RNAs with a length of 20-22 nucleotides. They bind specifically to target genes, thereby inducing the degradation of target gene mRNAs or inhibiting their translation, negatively regulating the expression of target genes at the post-transcriptional level, and thus affecting the biological characteristics of cells. Studies have shown that some miRs play an important role in controlling inflammation and can provide a good microenvironment for periodontal tissue regeneration. However, miRs are unstable molecules with relatively high molecular weight and negative charge, which makes it difficult for them to penetrate the cell membrane. Therefore, the effect of transfection of miRs alone is poor and it is impossible to apply it in clinical practice. In addition, the current transfection reagents are expensive and have low transfection efficiency in tissues. For example, using liposome transfection cannot effectively use miRs in clinical practice.

[0004] In summary, it is necessary to explore miRs with periodontal anti-inflammatory effects and modify them so that they can penetrate the cell membrane and improve their transfection effect, thereby playing an effective anti-inflammatory role in periodontal clinical treatment. Summary of the invention

[0005] In order to overcome the deficiencies of the above-mentioned prior art, the present invention designs a HA-PEI-miR-146a composite material, which utilizes HA-PEI to enable HA-PEI to successfully carry miR-146a into cells without being toxic to the cells, thereby enabling miR-146a to play a role in inhibiting inflammation in the clinical treatment of periodontal disease.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides a method for preparing a HA-PEI-miR-146a nanocomposite material, the method comprising the following steps:

[0008] S1. Synthesis of HA-PEI: First, dissolve NHS and EDC in water, then add NaCl solution containing PEI, stir at 50-70°C for 20-40 minutes, then add NaCl solution containing HA, continue stirring for 20-30 hours, and dialyze after the reaction is completed to obtain HA-PEI;

[0009] S2. Mix the HA-PEI prepared in step S1 with the miR-146a mimic in phosphate buffered saline and incubate at room temperature for 7-15 minutes.

[0010] Preferably, the dosage ratio of HA-PEI and miR-146a mimics in S2 is 50-60 mL: 0.9-1 mg.

[0011] Preferably, the solvent of the phosphate buffered saline in S2 is PBS, and the pH value is 7.0-8.0.

[0012] Preferably, the mass ratio of NHS, EDC, PEI and HA in S1 is 20-24:33-37:13-17:90-110.

[0013] Preferably, the volume ratio of the aqueous solution of NHS and EDC, the NaCl solution containing PEI and the NaCl solution containing HA in S1 is 2:1:1.

[0014] Preferably, the relative molecular weight of the PEI in S1 is 10,000 and the purity is 99%.

[0015] Preferably, the concentration of the NaCl solution in S1 is 0.4-0.6M.

[0016] Preferably, the dialysis in S1 is performed until the conductivity of the product is consistent with the conductivity of pure water.

[0017] The second aspect of the present invention provides a HA-PEI-miR-146a nanocomposite material prepared by the preparation method described in the first aspect.

[0018] The third aspect of the present invention provides use of the HA-PEI-miR-146a nanocomposite material described in the second aspect in the preparation of a drug for treating periodontitis.

[0019] The HA-PEI-miR-146a composite material constructed by the method of the present invention utilizes the characteristics of HA-PEI such as reduced electric potential, easy cell endocytosis, good stability and targeting, low toxic side effects, high biocompatibility, and reduced phagocytosis of the immune system in the body, and is combined with miR-146a having an anti-inflammatory effect, so that HA-PEI can successfully carry miR-146a into cells without being toxic to the cells, thereby enabling miR-146a to play a role in inhibiting inflammation in clinical periodontal treatment.

[0020] Preferably, in order to enrich the application forms of the drug and make it suitable for different ranges, the drug also includes a pharmaceutically acceptable carrier.

[0021] Furthermore, the carrier is an acceptable functional pharmaceutical excipient in the pharmaceutical field, including surfactants, suspending agents, emulsifiers and some new pharmaceutical polymer materials, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid polylactic acid copolymer (PLGA), hyaluronic acid, etc. It may also include excipients such as diluents, adhesives, lubricants, disintegrants, cosolvents, stabilizers, etc.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention has previously found that miR-146a has an obvious periodontal anti-inflammatory effect (Xie YF, Shu R, Jiang SY, Song ZC, Guo QM, Dong JC, Lin ZK. miRNA-146 negatively regulates the production of pro-inflammatory cytokines via NF-κB signaling in human gingival fibroblasts. J Inflamm (Lond), 2014, 11 (1): 38.; Jiang SY, Xue D, Xie YF, Zhu DW, Dong YY, Wei CC, Deng JY. The negative feedback regulation of microRNA-146a in human periodontal ligament cells after Porphyromonas gingivalis lipopolysaccharide stimulation. Inflamm Res, 2015, 64 (6): 441-451.), but because miR-146a is difficult to penetrate the cell membrane, the simple transfection effect is poor, and the use of liposome transfection cannot be effectively realized in clinical use. For this reason, the present invention designs a preparation method of a HA-PEI-miR-146a composite material, which utilizes the characteristics of HA-PEI with weakened potential, easy cell endocytosis, good stability and targeting, low toxicity and side effects, high biocompatibility, and reduced phagocytosis of the immune system in the body, and combines it with miR-146a with anti-inflammatory effect, and improves the cell permeability of miR-146a with the help of HA-PEI. Further research found that the HA-PEI-miR-146a nanocomposite material of the present invention has the advantages of easy transfection and good transfection effect, can give full play to the anti-inflammatory effect of miR-146a on periodontium, and if it can be applied to the clinical practice of periodontal treatment, it is cheap, conducive to widespread use, and meets the actual clinical needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of cytotoxicity test of HA-PEI-miR-146a nanocomposite are shown;

[0025] Figure 2 Fluorescence images of cells transfected with Cy3-labeled HA-PEI-miR-146a nanocomposite;

[0026] Figure 3is the mRNA expression level of IL-6, IL-8 and TNF-α in different experimental groups. DETAILED DESCRIPTION

[0027] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0028] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0029] Example 1: Preparation of HA-PEI-miR-146a nanocomposite

[0030] (1) Synthesis of HA-PEI: 0.2900 g NaCl was dissolved in 10 mL deionized water to prepare a 0.5 M NaCl solution for use. Then, 0.0220 g NHS (N-hydroxysuccinimide) and 0.0350 g EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were dissolved in 10 mL deionized water, and the resulting solution was transferred to a round-bottom flask. Then, 5 mL of a 0.5 M NaCl solution containing 0.0150 g PEI (polyetherimide, relative molecular weight of 10,000, 99%) was transferred to the above flask. After stirring in a 60° C. water bath for 30 min, 5 mL of a 0.5 M NaCl solution containing 0.1000 g HA (hyaluronic acid) was transferred to the above flask, and stirring in a 60° C. water bath was continued for 24 h. After the reaction is completed, the reactants are placed in a dialysis bag (molecular weight cutoff (MWCO) is 1000) and dialyzed with pure water until the conductivity of the water in the dialysis water tank is consistent with the conductivity of pure water, indicating that the dialysis is completed. Finally, the dialyzed product is freeze-dried for 3 days for later use.

[0031] (2) Preparation of HA-PEI-miR-146a nanocomposite materials: HA-PEI (50 mg / mL): miR-146a mimic (20 μM, purchased from Ruibo Bio; the sequence of miR-146a is recorded in NCBI: Accession: NR_029701.1): α-MEM culture medium were mixed in a volume ratio of 1.5:5:50, and incubated in an incubator for 30 min before use.

[0032] Example 2: Detection of toxicity and anti-inflammatory effects of HA-PEI-miR-146a nanocomposite

[0033] (1) Cytotoxicity detection of HA-PEI-miR-146a nanocomposites

[0034] Gingival fibroblasts HGF-1 were plated in 96-well plates and grouped according to the experimental purpose: (1) control group; HA-PEI-scramble RNA (purchased from Ruibo Biotechnology, as a negative control); (2) HA-PEI-miR-146a group.

[0035] Five replicate wells were set up in each group, and osteogenic induction medium [containing 10% fetal bovine serum, 1.5% human-related stem cell osteogenic differentiation supplement (human-related stem cell osteogenic differentiation kit, purchased from: China Saiye Biotechnology Co., Ltd.), 1% double antibody-containing cell basal medium] was added and incubated overnight in a 37°C incubator. After the cells adhered to the wall, 2.5 μL of HA-PEI-miR-146a or HA-PEI-scramble RNA nanocomposite materials were added according to the above experimental groups, and the cells were continued to be cultured in the incubator. CCK-8 reagent was added at the same time at 0d, 24h, 3d, 5d, and 7d, and the OD value of each well at a wavelength of 450nm was measured with an enzyme marker.

[0036] Figure 1 The experimental results showed that there was no statistical difference in OD values ​​between different groups at the same time (P<0.05), indicating that the HA-PEI-miR-146a nanocomposite material had no cytotoxicity.

[0037] (2) Detection of the anti-inflammatory effect of HA-PEI-miR-146a nanocomposite

[0038] A 6-well plate was routinely plated, and the culture medium was discarded when the gingival fibroblasts grew to about 80%, and the cells were replaced with medium. 56.5 μL of nanocomposite materials (Cy3-labeled HA-PEI-miR-146a nanomaterials) were used for transfection in the transfection group, and the transfection effect was observed by fluorescence microscopy after culture. At the same time, samples were collected on day 21 to extract total cell RNA, reverse transcribed to synthesize cDNA, and then the expression of inflammatory factors (IL-6, IL-8, TNF-α) was detected by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The groups in the inflammatory factor detection experiment were: PC group: non-transfection stimulation group; HA-PEI / SC group: transfection of HA-PEI-scrambleRNA; HA-PEI / miR-146a group: transfection of HA-PEI-miR-146; LPS+HA-PEI / SC group: transfection of HA-PEI-scrambleRNA+1μg / mL Pg LPS stimulation; LPS+HA-PEI / miR-146a group: transfection of HA-PEI-miR-146a+1μg / mL Pg LPS stimulation.

[0039] Figure 2 The experimental results show that HA-PEI-miR-146a nanocomposite has a high transfection effect on cells. At the same time, the research results ( Figure 3 ) showed that compared with the PC group and HA-PEI / SC group, the relative expression of inflammatory factors in the HA-PEI / miR-146a group was reduced, but there was no statistical difference; however, the expression of IL-6, IL-8, and TNF-α in the LPS+HA-PEI / miR-146a group was significantly lower than that in the LPS+HA-PEI / SC group (P<0.05). The above results indicate that HA-PEI / miR-146a inhibits the inflammatory microenvironment produced by Pg LPS. The above results indicate that HA-PEI-miR-146a can inhibit the expression of inflammatory factors produced by Pg LPS and produce a microenvironment that is conducive to tissue repair.

[0040] In summary, the HA-PEI-miR-146a nanocomposite material prepared by the present invention has the advantages of easy transfection and good transfection effect, can give full play to the anti-inflammatory effect of miR-146a on periodontal tissues, and if it can be applied to the clinical practice of periodontal treatment, it is cheap, conducive to widespread use, and meets the actual clinical needs.

[0041] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A method for preparing a HA-PEI-miR-146a nanocomposite material, characterized in that: The following steps are involved: S1. Synthesis of HA-PEI: First, dissolve NHS and EDC in water, then add NaCl solution containing PEI, stir at 50-70°C for 20-40 minutes, then add NaCl solution containing HA, continue stirring for 20-30 hours, and dialyze after the reaction is completed to obtain HA-PEI; S2. Mix the HA-PEI prepared in step S1 with the miR-146a mimic in phosphate buffered saline and incubate at room temperature for 7-15 minutes.

2. The method for preparing a HA-PEI-miR-146a nanocomposite material according to claim 1, characterized in that: The dosage ratio of HA-PEI and miR-146a mimics described in S2 is 50-60mL:0.9-1mg.

3. The method for preparing a HA-PEI-miR-146a nanocomposite material according to claim 1, characterized in that: The solvent of the phosphate buffered saline in S2 is PBS, and the pH value is 7.0-8.

0.

4. The method for preparing a HA-PEI-miR-146a nanocomposite material according to claim 1, characterized in that: The mass ratio of NHS, EDC, PEI and HA described in S1 is 20-24:33-37:13-17:90-110.

5. The method for preparing a HA-PEI-miR-146a nanocomposite material according to claim 1, characterized in that: The volume ratio of the aqueous solution of NHS and EDC, the NaCl solution containing PEI and the NaCl solution containing HA in S1 is 2:1:

1.

6. The method for preparing a HA-PEI-miR-146a nanocomposite material according to claim 1, characterized in that: The relative molecular weight of PEI described in S1 is 10000 and the purity is 99%.

7. The method for preparing a HA-PEI-miR-146a nanocomposite material according to claim 1, characterized in that: The concentration of the NaCl solution in S1 is 0.4-0.6M.

8. The method for preparing a HA-PEI-miR-146a nanocomposite material according to claim 1, characterized in that: The dialysis described in S1 is performed until the conductivity of the product is consistent with the conductivity of pure water.

9. The HA-PEI-miR-146a nanocomposite material prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the HA-PEI-miR-146a nanocomposite material according to claim 9 in preparing a drug for treating periodontitis.

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