阅读说明：本技术 一种纳米颗粒及其制备方法和应用 (Nano-particles and preparation method and application thereof ) 是由 胡涛 张月 雷蕾 田雨婷 胥一尘 于 2021-09-30 设计创作，主要内容包括：本发明公开了一种纳米颗粒及其制备方法和应用,涉及龋病防治领域。本发明提供了一种抗龋的新的纳米材料,其包括携带变异链球菌vicR基因反义RNA的重组质粒和包覆载体,该重组质粒可以调控变异链球菌vicR基因的表达,可以降低vicR基因的表达水平,减少变异链球菌胞外多糖的合成,降低其生物膜的黏附聚集能力,通过转录后微调控的方式达到抗龋作用。重组质粒与包覆载体通过静电吸附结合。包覆载体由价格低廉且生物相容性好的精胺改性的淀粉材料制得。由于重组质粒带负电荷,而淀粉表面电荷为负,发明人通过精胺对淀粉材料进行表面改性,以使得淀粉的表面均带有正电荷,从而满足了重组质粒与精胺改性后的淀粉材料的静电结合。(The invention discloses a nano particle and a preparation method and application thereof, and relates to the field of caries prevention and treatment. The invention provides a novel anti-caries nano material, which comprises a recombinant plasmid and a coating vector, wherein the recombinant plasmid carries streptococcus mutans vicR gene antisense RNA, can regulate and control the expression of streptococcus mutans vicR gene, can reduce the expression level of the vicR gene, reduces the synthesis of streptococcus mutans extracellular polysaccharide, reduces the adhesion and aggregation capability of a biological membrane, and achieves the anti-caries effect in a post-transcriptional micro-regulation mode. The recombinant plasmid and the coated carrier are combined through electrostatic adsorption. The coating carrier is prepared from spermine modified starch material with low price and good biocompatibility. Because the recombinant plasmid is negatively charged and the surface charge of the starch is negative, the inventor modifies the surface of the starch material through spermine to ensure that the surface of the starch is positively charged, thereby satisfying the electrostatic combination of the recombinant plasmid and the starch material modified by the spermine.)

1. A nanoparticle, which comprises a coating carrier and a recombinant plasmid carrying antisense RNA of a vicR gene of streptococcus mutans, wherein the recombinant plasmid and the coating carrier are combined through electrostatic adsorption, and the material of the coating carrier is spermine modified starch material.

2. The nanoparticle according to claim 1, wherein the mass ratio of the recombinant plasmid to the coated carrier is 1: 2-10;

preferably, the mass ratio of the recombinant plasmid to the coated vector is 1: 8.

3. The nanoparticle according to claim 2, wherein the spermine-modified starch material is: the product of the reaction of the starch treated by the N, N' -carbonyl diimidazole and spermine; the starch is prepared by enzymolysis of amylase.

4. The nanoparticle according to claim 1, wherein the recombinant plasmid of antisense RNA to the VicR gene of Streptococcus mutans is pDL278 AsvicR.

5. A process for the preparation of nanoparticles according to any one of claims 1 to 4, characterized in that it comprises: mixing spermine-modified starch material with the recombinant plasmid.

6. The method according to claim 5, wherein the nanoparticles are prepared by using an emulsion-bidirectional method for preparing the spermine-modified starch material and the recombinant plasmid; it includes: mixing a spermine modified starch material and the recombinant plasmid to prepare a dispersed phase, taking polyethylene glycol as a continuous phase, mixing the dispersed phase and the continuous phase, and collecting a precipitate;

preferably, the mixing volume ratio of the dispersed phase to the continuous phase is 1: 4-8;

preferably, the concentration of the polyethylene glycol in the continuous phase is 100g-600g/L, and the molecular weight of the polyethylene glycol is 8000-20000;

preferably, the collecting the precipitate comprises: mixing the dispersed phase with the continuous phase, standing, and centrifuging to collect precipitate; preferably, the precipitate is collected and washed with ethanol.

7. The method of claim 5, further comprising preparing a spermine-modified starch material comprising: firstly, mixing N, N' -carbonyldiimidazole with starch subjected to enzymolysis by amylase for reaction, and then mixing a reaction product with spermine for reaction;

preferably, the mixing reaction time of the N, N' -carbonyldiimidazole and starch after amylase enzymolysis is 2-3 h; the reaction product and spermine are mixed and reacted for 18-24h at 35 +/-2 ℃ in an oil bath; preferably, after mixing and reacting with spermine, dialysis and freeze drying are sequentially performed.

8. The preparation method according to claim 7, wherein the molar mass ratio of the N, N' -carbonyldiimidazole to glucose units in starch is 1: 2-6;

preferably, the molar mass ratio of the spermine to glucose units in the starch is 1: 2-6;

preferably, the molar ratio of spermine to N, N' -carbonyldiimidazole is 5-1: 1;

preferably, the starch is starch treated by pullulanase and alpha-amylase in sequence.

9. Use of the nanoparticles according to any one of claims 1 to 4 or the nanoparticles prepared by the preparation method according to any one of claims 5 to 8 in an oral disease control drug or a bacteriostatic drug.

10. Use according to claim 9, wherein the oral disease is caries, plaque, gum bleeding or tartar.

Technical Field

The invention relates to the field of caries prevention and treatment, and particularly relates to nanoparticles and a preparation method and application thereof.

Background

Caries, commonly known as dental caries and decayed tooth, is a bacterial disease which can cause secondary pulpitis and periapical periodontitis, and even inflammation of alveolar bone and jaw bone. If not treated in time, the lesion may form a cavity. Caries is characterized by high incidence rate and wide distribution, and is a common disease of oral cavity. Caries occurs in close association with the formation of plaque biofilm.

The currently accepted etiology of caries is the four-in-one theory, which mainly involves bacteria, oral environment, host and time. The basic points are as follows: cariogenic dietary sugar adheres only to the tooth surface, an acquired film formed by salivary proteins. The acquired film can be firmly attached to the tooth surface, and can generate acid in the deep layer of bacterial plaque at proper temperature and enough time to attack the tooth, so that the tooth is demineralized, and organic matter is further destroyed to generate a cavity.

Streptococcus mutans is the main cariogenic bacterium closely related to the development of caries. The oral bacterial liquid can inhibit the growth of other symbiotic bacteria by metabolizing carbohydrate in the oral cavity to produce acid and reducing the pH value of dental plaque biomembrane, thereby laying the predominance of the oral bacterial liquid in the biomembrane. Mutans streptococci rely on the protection of the extracellular matrix and other signaling pathways to cope with the stress of environmental changes through biofilm formation. The secreted glucosyltransferase and fructosyltransferase can effectively synthesize extracellular polysaccharide, promote adhesion of bacteria and tooth surfaces on one hand, and provide binding sites for colonization of other bacteria on the other hand, thereby promoting formation of stable biomembranes. The main cariogenic virulence factors of streptococcus mutans mainly include acid production, acid tolerance, exopolysaccharide synthesis, biofilm formation and the like.

At present, the problem of poor anti-caries effect exists in caries prevention and treatment.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The present invention aims to provide a nanoparticle, a method for preparing the same, and an application thereof to solve the above technical problems.

The invention is realized by the following steps:

the invention provides a nanoparticle, which comprises a coating carrier and a recombinant plasmid carrying streptococcus mutans vicR gene antisense RNA, wherein the recombinant plasmid and the coating carrier are combined through electrostatic adsorption, and the coating carrier is made of a spermine modified starch material.

The inventor provides a novel anticarious nanomaterial, which comprises a recombinant plasmid carrying streptococcus mutans vicR gene antisense RNA, wherein the recombinant plasmid can regulate and control the expression of streptococcus mutans vicR gene, can reduce the expression level of vicR gene, reduces the synthesis of extracellular polysaccharide of streptococcus mutans, reduces the adhesion and aggregation capability of a biological membrane of streptococcus mutans, and achieves an anticarious effect in a post-transcriptional micro-regulation mode.

Electrostatically associated with the recombinant plasmid is a coated vector. The coating carrier is prepared from spermine modified starch material with low price and good biocompatibility. Because the recombinant plasmid is negatively charged and the surface charge of the starch is negative, the inventor modifies the surface of the starch material through spermine to ensure that the surface of the starch is positively charged, thereby satisfying the electrostatic combination of the recombinant plasmid and the starch material modified by the spermine.

The combination of starch and spermine is a material combination which is screened out by the inventor through long-term exploration and a large number of tests and has good biocompatibility, easy decomposition and high safety performance, and has special technical effects.

Starch is a common macromolecule in nature, widely exists in various foods, is low in price and is easy to obtain. When the starch is used for preparing the nano-particles for treating or preventing the caries, amylase in saliva can be conveniently degraded to expose the connected recombinant plasmid, so that the growth speed of cariogenic bacteria is reduced, the expression quantity of VicR protein is reduced, the biofilm formation capability and the adhesion capability of bacteria are inhibited, and the synthesis of streptococcus mutans extracellular polysaccharide is reduced.

Spermine is a polyamine substance, which is widely present in bacteria and most animal cells, is an important substance for promoting cell proliferation, and has good biocompatibility.

In a preferred embodiment of the present invention, the mass ratio of the recombinant plasmid to the coated vector is 1: 2-10. The inventors found that the binding ability of the spermine-modified starch material to the plasmid is at a higher level within the above mass ratio range.

In an alternative embodiment, the mass ratio of recombinant plasmid to coated vector is 1: 8. When the mass ratio of the recombinant plasmid to the coated vector is 1:8, the microspheres prepared at the ratio of 1:8 are found to have a good morphology when an emulsion bidirectional method is used.

In a preferred embodiment of the present invention, the spermine modified starch material is: the product of the reaction of the starch treated by the N, N' -carbonyl diimidazole and spermine; the starch is prepared by enzymolysis of amylase. N, N' -carbonyldiimidazole is used as an activator to introduce groups into the hydroxyl groups of the starch.

In a preferred embodiment of the present invention, the recombinant plasmid of the antisense RNA of the streptococcus mutans vicR gene is pDL278 AsvicR. The recombinant plasmid is constructed by a patent CN108130300B, the construction method is the same as that of the patent CN108130300B, and the streptococcus mutans is CCTCCM 2017746.

The invention provides a preparation method of nanoparticles, which comprises the following steps: mixing the spermine modified starch material with the recombinant plasmid.

In the preferred embodiment of the invention, the spermine modified starch material and the recombinant plasmid are prepared into the nano-particles by an emulsion bidirectional method; it includes: mixing spermine modified starch material and recombinant plasmid to prepare a dispersed phase, taking polyethylene glycol as a continuous phase, mixing the dispersed phase and the continuous phase, and collecting precipitate;

in an alternative embodiment, the mixing volume ratio of the dispersed phase to the continuous phase is from 1:4 to 8. When the volume ratio of the dispersed phase to the continuous phase is 1:8, the combination of the spermine modified starch material and the plasmid has stronger capacity of forming microspheres.

In an alternative embodiment, the concentration of polyethylene glycol in the continuous phase is from 100g to 600g/L, and the molecular weight of polyethylene glycol is 8000-.

In an alternative embodiment, collecting the precipitate comprises: mixing the dispersed phase and the continuous phase, standing, and then centrifuging and collecting precipitate; in an alternative embodiment, the precipitate is collected and washed with ethanol. Optionally, the ethanol washing step is repeated.

The standing is carried out at 0-8 deg.C for more than 24 hr. The precipitate was collected more thoroughly by standing.

In a preferred embodiment of the present invention, the above preparation method further comprises the preparation of a spermine-modified starch material, which comprises: firstly, mixing N, N' -carbonyl diimidazole with starch subjected to enzymolysis by amylase for reaction, and then mixing a reaction product with spermine for reaction.

In an alternative embodiment, the mixing reaction time of the N, N' -carbonyldiimidazole and the starch after the amylase enzymolysis is 2-3 h; the reaction product and spermine are mixed and reacted for 18 to 24 hours at 35 +/-2 ℃ in an oil bath; in an alternative embodiment, the powder is prepared by mixing and reacting with spermine, and then dialyzing and freeze-drying the mixture in sequence.

In a preferred embodiment of the present invention, the molar mass ratio of the N, N' -carbonyldiimidazole to glucose units in the starch is 1: 2-6. For example, it may be 1:2, 1:4, 1:5 or 1: 6.

The inventors found that when the molar mass ratio of spermine to glucose units in the starch is 1:2-6, the surface of the spermine-modified starch material is positively charged. The spermine modified starch material has a higher zeta potential when the molar mass ratio is 1: 2.

In an alternative embodiment, the molar ratio of spermine to N, N' -carbonyldiimidazole is 5-1: 1.

In an alternative embodiment, the starch is a starch treated with pullulanase and α -amylase in that order. The pullulanase can specifically cut alpha-1, 6 glycosidic bonds in the branch point of the amylopectin, and cut off the whole branch structure to form amylose. Alpha-amylases can hydrolyze the alpha-1, 4 glucosidic bonds in starch to form a starch of smaller molecular weight.

The invention also provides application of the nano-particles or the nano-particles prepared by the preparation method in oral disease prevention and treatment medicines or bacteriostatic medicines.

In preferred embodiments of the invention, the oral diseases include, but are not limited to, caries, plaque, gum bleeding, or tartar. Bacteriostatic agents include, but are not limited to, acidogenic and gram-positive bacteria; acid-producing bacteria including but not limited to Streptococcus mutans, Actinomyces, and Lactobacillus are within the scope of the invention, provided that they are capable of producing acid by carbohydrate decomposition, resulting in demineralization of tooth mineral.

The invention has the following beneficial effects:

the invention provides a novel anticarious nanomaterial, which comprises a recombinant plasmid carrying streptococcus mutans vicR gene antisense RNA, wherein the recombinant plasmid can regulate and control the expression of streptococcus mutans vicR gene, can reduce the expression level of the vicR gene, reduce the synthesis of streptococcus mutans extracellular polysaccharide, reduce the adhesion and aggregation capability of a biological membrane of streptococcus mutans extracellular polysaccharide, and achieve anticarious effect in a post-transcriptional micro-regulation mode.

Electrostatically associated with the recombinant plasmid is a coated vector. The coating carrier is prepared from spermine modified starch material with low price and good biocompatibility. Because the recombinant plasmid is negatively charged and the surface charge of the starch is negative, the inventor modifies the surface of the starch material through spermine to ensure that the surface of the starch is positively charged, thereby satisfying the electrostatic combination of the recombinant plasmid and the starch material modified by the spermine.

The anti-caries novel nano material provided by the invention has good application prospect for development of oral disease prevention and treatment medicines or antibacterial medicines.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a flowchart of the process for synthesizing spermine-modified starch material combined with a target plasmid to form a microsphere material and a scanning electron microscope image of the microsphere material in examples 1 and 3 of the present invention;

FIG. 2 is a schematic diagram showing Zeta potential results, agarose gel electrophoresis results and spermine modification in the binding ability of the spermine-modified starch material to plasmids in Experimental example 1 of the present invention;

FIG. 3 is a schematic diagram of the agarose gel electrophoresis results of the binding ability of spermine-modified starch material and plasmid in Experimental example 2, the agarose gel electrophoresis results of the release and protection of microsphere material to plasmid in Experimental example 2, and the enzymolysis;

FIG. 4 is a graph showing the results of cytotoxicity test and cck8 of the microsphere material according to Experimental example 3 of the present invention, the results of introducing plasmids into Streptococcus mutans and expressing GFP in the microsphere material according to Experimental example 4 of the present invention, and the mode of action of the microsphere material;

FIG. 5 shows the results of the detection of the expression contents of the vicR and dexA proteins of Streptococcus mutans by the SMS-AS microsphere material in Experimental example 5 of the present invention, and the results of the detection of the expression of virulence related genes of Streptococcus mutans by the SMS-AS microsphere material in Experimental example 6 of the present invention;

FIG. 6 shows the result of testing the influence of microsphere material on the synthesis of extracellular polysaccharide of Streptococcus mutans in Experimental example 7;

FIG. 7 shows the results of fluorescence-staining and laser-confocal detection of the effect of microsphere materials on the synthesis of extracellular polysaccharides of Streptococcus mutans in Experimental example 7;

FIG. 8 is a flow chart showing in vivo results of studies on influence of microsphere materials on cariogenicity of Streptococcus mutans in Experimental example 8 of the present invention, gene sequencing results in Experimental example 9 of the present invention, and animal experiments.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The present embodiment provides a method for synthesizing spermine modified Starch Material (SMS), which specifically includes the following steps performed in sequence, as shown in fig. 1:

(1) firstly, carrying out enzymolysis on macromolecular corn starch: 20g of corn starch (corn starch, Solebao, China) was dissolved in 400ml of PBS buffer to prepare a 5% starch solution, and the solution was placed in an oil bath pan at 80 ℃ and heated for 30 minutes to gelatinize the starch. Cooling the gelatinized starch to 60 ℃ at room temperature, adding 1g of pullulanase (pullulanase, Meclin, China), and reacting for 14h at 60 ℃ in an oil bath. Heating to 95 deg.C, and maintaining for 30min to inactivate pullulanase. Cooling the starch solution to 60 ℃ at room temperature, adding 0.1g of alpha-amylase, carrying out oil bath reaction at 60 ℃ for 30 minutes, heating to 95 ℃, and keeping for 30 minutes to inactivate the amylase. The solution was cooled to room temperature, concentrated by centrifugation at 10000rpm for 10min and dialyzed against deionized water for 3 days (molecular weight cut-off of 7000). Freeze drying the starch solution, and placing the powder in a dryer for later use.

(2) Synthesis of spermine modified Starch Molecules (SMS): and (2) dissolving the corn starch subjected to enzymolysis in the step (1) in anhydrous dimethyl sulfoxide to prepare a solution with the concentration of 20 mg/ml. N, N' -Carbonyldiimidazole (CDI) was added in various ratios (the molar mass ratio of CDI to glucose units in starch was 1:2, 1:4 and 1:6) and reacted at room temperature for 2 hours. Spermine (mol)/cdi (mol) ═ 1:1 with molar mass of spermine to glucose units of 1:2, 1:4, 1:6) was added in different molar ratios and reacted in an oil bath at 35 ℃ for 24 h. The reaction product was dialyzed against deionized water for 3 days (molecular weight cut-off 7000), freeze-dried, and the powder was placed in a desiccator for use.

Example 2

This example provides a method for extracting recombinant plasmids carrying the S.mutans antisense vicR and GFP genes.

The recombinant plasmid is derived from Escherichia coli glycerobacteria (with the preservation number of CCTCC NO: M2017746) preserved in the preliminary experiment of the subject group, cultured for 48h by using LB solid medium added with spectinomycin (spe), then single colony is picked up, and cultured for 16-20h in a constant temperature shaking table (37 ℃, 300rpm) by using LB liquid medium added with spectinomycin (spe). The obtained bacterial liquid was extracted into plasmid (Tiangen, China) using Tiangen endotoxin-free large extraction kit.

Example 3

This example provides a method for the synthesis of spermine modified starch material plasmid-bound microspheres (SMS-AS).

Synthesizing the spermine modified starch material plasmid-bonded microspheres (SMS-AS) by an emulsion two-way method.

1. Preparation of the continuous phase: dissolving polyethylene glycol 20000 in deionized water (4g/10ml), heating in water bath at 80 deg.C for one hour to dissolve completely, subpackaging PEG20000 solution in centrifuge tube, and heating in water bath at 50 deg.C for use.

2. Preparation of the dispersed phase: the spermine modified starch material prepared in example 1 was dissolved in sterile deionized water to prepare a spermine modified starch solution (50mg/ml), and the spermine modified starch solution was subjected to water bath at 50 ℃ for 20 minutes and ultrasonic treatment for 5 minutes to fully disperse the spermine modified starch molecules. The plasmid carrying ASvicR prepared in example 2 was mixed with the spermine modified starch solution at a mass ratio of 1:8, shaken for 1 minute to mix thoroughly to prepare a dispersed phase.

3. Pouring the dispersed phase into the continuous phase at a volume ratio of 1:8, fully inverting and uniformly mixing, and oscillating for 10 minutes to fully disperse the dispersed phase. The resulting liquid was placed in a refrigerator at 4 ℃ for over 24 hours, centrifuged at 5000rpm for 15 minutes, and the supernatant was decanted. Washing the precipitate with 75% ethanol for 3 times, dehydrating the precipitate with anhydrous ethanol, and placing the product in an oven at 50 deg.C overnight to obtain white powder (SMS-ASvicR nanoparticle material, namely SMS-AS), and placing in a dryer.

The scanning electron microscope images of the prepared microsphere nano material in ethanol and deionized water are shown as B in FIG. 1.

Experimental example 1

The experimental example tests the binding capacity of spermine modified starch materials with different proportions and plasmids.

The Zeta potential of three spermine modified starch materials was tested using a Malvern nanometer particle size potentiometer at molar mass ratios of spermine to glucose units of 1:2, 1:4, 1:6, respectively, at which time the molar mass ratio of CDI to glucose units in the starch was controlled to be 1: 2.

As a result, as shown in fig. 2 a, the surface charge of the starch material is negative, and after the starch material is modified with spermine, the surface of the modified material is positively charged. And, of the three spermine modified starch materials with spermine/glucose units of 1:2, 1:4, 1:6, the material of group 1:2 has a higher zeta potential. The surface of the spermine modified starch material is positively charged, and after the spermine modified starch material and the target plasmid are synthesized into the microsphere in a ratio of 1:2, the surface charge of the SMS-AS microsphere material is negative, which indicates that the spermine modified starch material and the target plasmid are fully combined.

Fig. 2B is an infrared spectrum of the spermine-modified starch material, and fig. 2C is an NMR spectrum of the spermine-modified starch material. Fig. 2D is the results of elemental analysis of three spermine-modified starch materials, and fig. 2E is a model diagram of spermine-modified starch materials.

Experimental example 2

The agarose gel electrophoresis method is used for evaluating the binding capacity of the spermine modified materials with different proportions and plasmids, 1% agarose gel is prepared, electrophoresis is carried out for 30 minutes at constant voltage of 100V, the specific result is shown as A in figure 3, and the spermine modified starch materials in the 1:2 group have the strongest binding capacity with the plasmids.

This example tests the release and protection of the microsphere material against plasmid.

(1) The release of plasmid was detected by electrophoresis in 1% agarose gel using alpha amylase for 30min on SMS-AS microsphere material prepared in example 1, at constant pressure of 100V for 30 min. As shown in FIG. 3B, the alpha-amylase can hydrolyze the alpha-1, 4 glycosidic bond in the SMS-AS microsphere material, so that the target plasmid is released.

(2) DNaseI is used for acting on the SMS-AS microsphere material and the plasmid alone for 15min, and the decomposition effect of DNaseI on the plasmid is detected by agarose gel electrophoresis at constant voltage of 100V for 30 min. As shown in FIG. 3C, the SMS-AS material has a protective effect on the desired plasmid.

Experimental example 3

This example tests the cytotoxicity test of the microsphere material and the cell viability of cck 8.

DMEM supplemented with 10% (v/v) fetal bovine serum, penicillin (100U/mL) and streptomycin (100. mu.g/mL) at 37 ℃ and 5% CO₂Culturing gingival fibroblast under the condition. (1) Cytotoxicity assay: cells in logarithmic growth phase were seeded in 96-well plates overnight at a density of 5000 cells per well. Cells were then treated with different concentrations (3.6, 14.4, and 28.8 μ g/mL) of SMS-ASvicR nanoparticles. After 24 hours of culture, Cell viability was evaluated using Cell Counting Kit-8(CCK8) Kit, and the results are shown in FIG. 4, B, C, wherein the results of CCK8 of the cells added with nanoparticles and the blank cells were not statistically different, and the Cell morphology was not significantly changed.

(2) Cell death and live staining: cells in logarithmic growth phase were seeded in 24-well plates overnight at a density of 20000 cells per well. After the cells had grown adherent under microscope observation, the cells were then treated with different concentrations (3.6, 14.4 and 18.8 μ g/mL) of SMS-ASvicR nanoparticles. After 24 hours of culture, cells were fluorescently labeled using the Calcein-AM/PI kit, and then observed under a fluorescence microscope, with the result shown in fig. 4A, the cells added with nanoparticles had no significant change in morphology compared to the blank cells.

Experimental example 4

In this example, the plasmid was successfully introduced into Streptococcus mutans using the microsphere material, and GFP protein was expressed.

Streptococcus mutans UA159 strain was inoculated to 5mLBHI, and after overnight resuscitation, the strain was diluted to 2mL of BHI medium containing 1% sucrose in a ratio of 1:20, and the medium was placed in a glass plate for culture. And (3) culturing in an anaerobic incubator until OD is 0.5, adding an SMS-AS microsphere material, culturing in the anaerobic incubator for 3h, and staining the cell wall of the streptococcus mutans by using Alexa Fluor 555. The GFP green fluorescent protein and cell wall in S.mutans were observed with confocal laser microscopy at excitation light levels of 555 and 488. As shown in FIG. 4D, S.mutans supplemented with SMS-AS material expressed green fluorescent protein, whereas the blank group did not. The schematic diagram is shown in fig. 4E.

Experimental example 5

In the experimental example, the SMS-AS microsphere material is used for detecting the expression content of the vicR and dexA proteins of the streptococcus mutans.

Extraction of total protein of streptococcus mutans: the streptococcus mutans UA159 strain is inoculated in 10mLBHI, after overnight recovery, diluted in 3mL BHI culture medium containing 1% sucrose according to the proportion of 1:10, cultured in a six-well plate, added with (SMS-AS, SMS, plasmid and PBS) every 4h, anaerobically cultured for 12h, scraped off a biomembrane, added with lysozyme and placed at 37 ℃ for 30min, ultrasonically crushed for 2min, centrifuged at 4 ℃ for 10min (4500 rpm), collected supernatant, subjected to nanodrop protein concentration determination, and added with protease inhibitor (Sigma) for later use. Western Blot validation of VicR protein:

running SDS-PAGE electrophoresis: protein gel (Precise 4-20% Tris-HEPES Gels; Waltham, MA, USA) sequence: SMS-AS, SMS, plasmid, PBS 160V isolation samples about 70 min. Secondly, film turning: voltage 70V and time 90 min. ③ Tris-HEPES protein gel with Coomassie blue staining, shaking table at room temperature overnight, decolorization. Closing 5 percent skim milk of the Nitrocellulose membrane, and placing the Nirocellulose membrane on a shaking table for 2 hours at room temperature. Incubation primary antibody: discarding the skim milk used for sealing, adding about 15mL of freshly prepared skim milk, adding 5 mu L of anti-VicR and anti-dexA monoclonal antibody, and placing on a shaking table for incubation for 2h at room temperature; milk was discarded and washed 3 times with TSBT for 10min each, approximately 15mL volume. Sixthly, incubation of the secondary antibody: about 15mL of freshly prepared skim milk was added, and 1.5. mu.L of anti-rabbitit secondary antibody was added at a dilution ratio of 1: 10000. Placing on a shaking table and incubating for 2h at room temperature; milk was discarded and washed 3 times with TSBT for 10min each, approximately 15mL volume. And seventh, developing: appropriate amounts of Immobilon Western (Milipore, USA) HRP substrate luminescence detection solution were prepared. About 200. mu.L of the prepared luminescence detection solution was uniformly applied to the NC membrane, and the NC membrane was wrapped in a clean safety film and left at room temperature in the dark for about 3min, and fixed to an Electrophoresis system (Bio-Rad, USA) in an X-ray film cassette. An X-ray film is taken out from the darkroom and placed on an NC film, and the darkbox is closed for about 10 min. Placing the exposed X-ray film in a developing solution developer prepared in advance for 3min, slightly washing with double distilled water, and placing in a fixing solution for 5 min.

The results are shown in fig. 5B, where the color of the vicR protein of the SMS-AS group is lighter and the color of the dexA protein is darker, indicating that the vicR protein of the streptococcus mutans group treated with the SMS-AS microsphere material is expressed in a lower amount and the dexA protein is expressed in a higher amount than the vicR protein of the PBS-treated streptococcus mutans group.

Experimental example 6

The detection of the SMS-AS microsphere material on the expression of the virulence related genes of the streptococcus mutans comprises the following steps:

the total RNA extraction method comprises the following steps: the Streptococcus mutans UA159 strain is inoculated in 10mLBHI, after overnight recovery, diluted in 3mL BHI culture medium containing 1% sucrose according to the proportion of 1:10, cultured in a six-well plate, added with (SMS-AS, SMS, plasmid and PBS) respectively every 4h, after anaerobic culture for 12h, scraped off a biological membrane, centrifuged to collect a bacterial sample, resuspended in PBS for standby, extracted with Masterpure RNA purification Kit (Epicentre), aspirated for 2 muL RNA sample, and applied with NanoDrop^TM2000c Spectrophotometer (Thermo Scientific, USA) measures the concentration and purity of RNA samples. Removal reaction of genomic DNA: mu.l of Turbo RNase-free DNase I (Ambion, Thermo Scientific, USA) was added to the obtained RNA solution, and the reaction was carried out at 42 ℃ for 2min to remove DNA remaining in the total RNA solution.

The reverse transcription method of bacterial mRNA comprises the following steps of taking the total RNA solution obtained in the above steps to perform reverse transcription reaction according to the method provided by RevertAID First Strand cDNA Synthesis (Thermo Scientific, USA) instruction: to a solution containing 1. mu.g of RNA were added in the following order: 1. mu.L Random Primer, 4. mu.L 5 × Reaction Buffer; 1 μ L of RiboLock RNase Inhibitor; 1 μ L RevertAid M-MuLV RT; after mixing 10. mu.L of RNase Free H2O by gentle vortex, the total volume was 20. mu.L. Acting at 25 deg.C for 5min, and at 42 deg.C for 60min, completing cDNA synthesis, and storing at-20 deg.C for use.

Real-time quantitative PCR: LightCycler 480SYBR Green I Master chimeric fluorescence method for Real-time PCR amplification. According to the method provided by LightCycler 480SYBR Green I Master (Roche, Basel, Switzterland) instructions, reaction system sample adding and two-step PCR amplification programs are carried out to carry out real-time PCR amplification on target genes, and Ct (cycle threshold) values of the target genes are obtained after amplification. In the experiment, a relative quantitative method is adopted, the relative expression level of a target gene is calculated and analyzed by a 2-delta Ct method, the expression difference of the streptococcus mutans biofilm genes using different materials (SMS-AS, SMS, plasmid and PBS) is obtained, and the bacterial gyrA gene is selected AS an internal reference. The experimental results were obtained by 3 independent real-time PCR amplification experiments, with the following RT-qPCR reaction system and primers used:

real-time PCR reaction System (20. mu.l)

Reagent	Amount of the composition used
		SYBR Green I Master	10μl
PCR Forward Primer(20uM)	1μl
		PCR Reverse Primer(20uM)	1μl
cDNA template	1μl
		Sterilized distilled water	7μl

Real-time PCR reaction primer sequence

The results are shown in fig. 5 a, and show that the vicR gene, gtfB gene, and gbpB gene of the streptococcus mutans group added with the SMS-AS nanomaterial are down-regulated, while the dexA gene is up-regulated.

Experimental example 7

This example explores the effect of microsphere materials on the synthesis of extracellular polysaccharides of Streptococcus mutans.

Adopting a scanning electron microscope detection method:

inoculating the streptococcus mutans UA159 strain to 10mLBHI, after overnight resuscitation, diluting the streptococcus mutans UA159 strain to 50mL of fresh BHI culture medium containing 1% sucrose according to a proportion of 1:10, adjusting OD value to enable OD600 to be approximately equal to 0.5, adding the bacterial suspension into a 12-hole plate, wherein each hole is 2mL, and each hole is added with 1 piece of sterile circular cover glass; each set of 3 wells was plated in parallel and (SMS-AS (3.6. mu.g), SMS-AS (14.4. mu.g), SMS-AS (28.8. mu.g) and PBS were added every 4 hours, and the 24-well plates were placed in an anaerobic incubator with a mixed gas (80% N2, 20% CO2) at 37 ℃ and incubated for 12 hours under standing. Carefully take out the round cover glass, shake wash the biomembrane 3 times with PBS buffer solution, suck the dry paper, stand for several minutes at room temperature, air-dry the biomembrane sample. Fixing the biological membrane slide in 2.5% glutaraldehyde, and placing at 4 ℃ in a dark place for 4 h; performing gradient dehydration with 30%, 50%, 75%, 85%, 95%, and 99% ethanol for 15min each time; displacement, drying of the metal spray, observation under a scanning electron microscope (observation Hillsboro, USA) and image acquisition were performed.

The results are shown in fig. 6, with increasing amounts of added SMS-AS nanoparticles, the biofilm bacteria were more sparsely packed around, and the extracellular matrix was more structurally thin, in a lesser proportion. The group to which 28.8 μ g of sms-AS nanomaterial was added had a significant reduction in extracellular matrix compared to the blank control group.

Meanwhile, a fluorescence staining and laser confocal detection method is adopted, and the method comprises the following steps:

inoculating the streptococcus mutans UA159 strain to 10mL of BHI, after overnight resuscitation, diluting the strain to 50mL of BHI culture medium containing 1% sucrose according to a proportion of 1:10, adjusting OD value to enable OD600 to be approximately equal to 0.5, adding the bacterial suspension into a 12-hole plate, wherein each hole is 2mL, and each hole is added with 1 piece of sterile circular cover glass; each group of 3 wells was in parallel, and (SMS-AS, SMS, plasmid, PBS) was added every 4h, and the 24-well plate was placed in an anaerobic incubator with a mixed gas (80% N2, 20% CO2) and incubated at 37 ℃ for 12 h. Carefully take out the round cover glass, shake wash the biomembrane 3 times with PBS buffer solution, suck the dry paper, stand for several minutes at room temperature, air-dry the biomembrane sample. When the biofilm was dried and wet, 50. mu.l of Syto 9 Nucleic Acid Stain (1:50 dilution) and Alexa were added dropwise to each biofilm slide647 (final concentration 1. mu.L/mL) fluorescence labeling of bacterial cells, room temperature incubation for 15 min; residual dye was washed off gently with PBS buffer and blotted dry with absorbent paper. When the biofilm was air-dried at room temperature to a wet state, the biofilm slide was placed on a slide, no fluorescence mounting oil mounting, and the sample was placed under a laser confocal microscope (TSP SP2, Leica, Solms, Germany) for observation as soon as possible.

The results are shown in fig. 7, where the streptococcus mutans biofilm structure using the SMS-AS nanomaterial group was loosely dispersed, and the biofilm thickness and extracellular matrix content were reduced compared to the blank control group.

Experimental example 8

This example was conducted to investigate in vivo the influence of microsphere materials on cariogenic properties of S.mutans.

After overnight resuscitation of the streptococcus mutans standard strain UA159, the culture was performed according to the following protocol 1:20 portions were diluted in 10mL of freshly prepared BHIS medium containing 1% sucrose, and the culture was continued for 3 hours to achieve an OD600 ≈ 0.5. Bacteria were spread on the surface of rat molar teeth using a sterile cotton swab and 0.2mL of bacterial solution was inoculated into the mandibular molar area of both rats once daily for one week continuously. Simultaneously, 0.2ml of equivalent material (SMS-AS, SMS, plasmid, PBS) was applied to the mandibular molar area of bilateral rats twice daily for two consecutive weeks, and diet was suspended 2h after treatment. Keyes2000# cariogenic diet (great success in Sichuan) and sterilized 5% sucrose solution were fed. Animals were kept for 2 weeks after treatment, rats were sacrificed by CO2 asphyxiation, jaw specimens were removed and washed 3 times with sterile PBS buffer. The samples were placed in 4% paraformaldehyde and fixed at 4 ℃ for 24 hours in the dark. And (3) using an ultrathin carborundum sheet to perform half-cutting along the near-far middle direction of the occlusal surface of the molar of the mandible under a cooling state, observing the caries condition under a stereoscopic microscope, and grading and scoring the smooth surface of the molar and the caries lesion of the pit and fissure of the rat according to a Keyes classical scoring method. According to the depth of the carious lesion, the method can be divided into: grade E caries lesions are limited to enamel; caries lesions of grade Ds are limited to enamel; dm caries lesions accumulate 1/4 and 3/4 more than dentin thickness; dx levels of caries lesions are accumulated to a depth exceeding the dentin thickness 3/4 or even the full thickness.

As shown in A, B in FIG. 8, the rats using the MS-AS nanomaterial group had lower dental caries rate in mandibular posterior molars and had the effect of inhibiting the occurrence of dental caries.

Experimental example 9

This example examined the safety of using microsphere materials by gene sequencing.

After three common oral cavity strains of streptococcus mutans standard strain UA159, streptococcus formaticus and streptococcus sanguis are revived overnight, the ratio of the standard strain UA159 to the standard strain formative strain to the standard strain haemophilus is 1:20 portions were diluted in 10mL of freshly prepared BHIS medium containing 1% sucrose, and the culture was continued for 3 hours to achieve an OD600 ≈ 0.5. Bacteria were spread on the surface of rat molar teeth using a sterile cotton swab and 0.2mL of bacterial solution was inoculated into the mandibular molar area of both rats once daily for three consecutive days. Saliva was taken from the rat mouth using a sterile swab. Then the nanometer material SMS-AS0.2ml is coated on the mandibular molar area of bilateral rats, and the diet supply is suspended 2h after the treatment. Saliva was taken from the rat mouth after 5h using a sterile swab.

The results of gene sequencing of rat saliva taken before and after the use of SMS-AS nanomaterial are shown in FIG. 8C, and there is no significant change before and after the use of SMS-AS nanomaterial.

In summary, the spermine modified starch material provided by the embodiment of the invention can regulate and control the vicR gene expression of streptococcus mutans by combining with plasmid microspheres (SMS-AS), and the expression level of VicR protein is reduced, so that the gene expression of dexA and the protein expression of dexA are increased, the biofilm formation capability is reduced, the content of extracellular polysaccharide of bacteria is reduced, and the related capability of caries is reduced.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.