Tooth-targeted nano drug-loaded micelle as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and relates to a tooth-targeted nano drug-loaded micelle, the tooth-targeted nano drug-loaded micelle comprises a copolymer micelle and a hydrophobic drug entrapped by the copolymer micelle, the copolymer is an alendronate modified amphiphilic block copolymer, and the hydrophobic drug is a hydrophobic antibacterial agent. The tooth-targeted nano drug-loaded micelle has a good antibacterial effect, HA adsorption performance and human wisdom tooth adsorption effect, is beneficial to selective delivery and accumulation of drugs in bone tissues, and realizes targeted drug delivery of teeth, so that the antibacterial effect of an antibacterial agent on bacteria causing caries is better exerted, and the tooth-targeted nano drug-loaded micelle has important significance on treatment and prevention of caries.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to a tooth-targeted nano-drug-loaded micelle, a preparation method thereof, and an application thereof. Background Art

[0002] Dental caries is a very common oral disease, and is called one of the three major prevention and treatment diseases of human beings together with tumors and cardiovascular diseases. Dental caries is mainly caused by Streptococcus mutans. Therefore, preventing and treating Streptococcus mutans is the key to treating oral dental caries. However, because the oral environment is complex, the prevention and treatment means are particularly important. At present, most of the treatment methods and drugs for dental caries cannot achieve a long-term antibacterial effect, and moreover, the drugs for treating dental caries do not have tooth-targeting properties; therefore, there is an urgent need to study a drug for preventing and / or treating dental caries that has tooth-targeting properties and a sustained and controlled release function.

[0003] In recent years, biodegradable polymer nanoparticles have attracted extensive attention because they can effectively increase the circulation time of drugs in the body, reduce the toxicity and side effects of the drugs themselves, improve the therapeutic effect of the drugs, and moreover, after the drug is released, the polymer nanoparticles will not accumulate in the body for a long time and can be excreted or absorbed after hydrolysis. The biodegradable polymer materials used as drug carriers can be classified into natural polymer materials, biosynthetic polymer materials, and synthetic polymer materials according to their sources; among them, due to the controllable structure and function of synthetic polymer materials, and the degradation rate in the body can be changed by adjusting the ratio and molecular weight of polymers, etc., they have become the main materials for researching and preparing polymer nanodrug carriers. Among them, poly(lactic acid-glycolic acid) copolymer (PLGA) has been approved by the US FDA for clinical application and is widely used in the application and research of drug delivery carrier materials.

[0004] In clinical practice, chlorhexidine, as a common disinfectant, is commonly used in the medical field for the disinfection of instruments, skin, and mucous membranes. In recent years, it has also been commonly used for oral anti-inflammatory and inhibiting the formation of dental plaque. Chlorhexidine has bactericidal and antibacterial effects on both Gram-positive and negative bacteria, and its drug efficacy is stronger than that of benzalkonium bromide, etc. More importantly, even in blood and serum, its drug efficacy is not affected, and it only inhibits bacterial spores. Therefore, chlorhexidine is a relatively ideal broad-spectrum bactericidal and antibacterial agent. Therefore, the present invention attempts to study a tooth-targeted nano-drug-loaded micelle with a sustained and controlled release function, which is expected to achieve long-term antibacterial. Summary of the Invention

[0005] Aiming at the problems and deficiencies existing in the prior art, the purpose of the present invention is to provide a tooth-targeted nano-drug-loaded micelle, a preparation method thereof, and an application thereof.

[0006] To achieve the purpose of the invention, the technical scheme adopted by the present invention is as follows:

[0007] In the first aspect of the present invention, a tooth-targeted nano-drug-loaded micelle is provided. The tooth-targeted nano-micelle comprises a copolymer micelle and a hydrophobic drug encapsulated by the copolymer micelle. The copolymer is an amphiphilic block copolymer modified with alendronate, and the hydrophobic drug is a hydrophobic antibacterial agent.

[0008] According to the above-mentioned tooth-targeted nano-drug-loaded micelle, preferably, the copolymer is PEG-PLGA in which the hydrophilic segment is modified with alendronate.

[0009] According to the above-mentioned tooth-targeted nano-drug-loaded micelle, preferably, in the PEG-PLGA in which the hydrophilic segment is modified with alendronate, the molecular weight of the PEG segment is 2000 Da, and the molecular weight of the PLGA segment is 20000 Da.

[0010] According to the above-mentioned tooth-targeted nano-drug-loaded micelle, preferably, the preparation method of PEG-PLGA in which the hydrophilic segment is modified with alendronate is as follows:

[0011] (1) Dissolve t-BOC-PEG-NHS in solvent A to obtain a t-BOC-PEG-NHS solution; drop the t-BOC-PEG-NHS solution into the alendronate solution, stir and react for 5 - 6 h after dropping, perform dialysis treatment on the reaction solution after the reaction, and perform lyophilization on the dialyzed reaction solution to obtain product A;

[0012] (2) Under the atmosphere of a protective gas, dissolve the product A in solvent B to obtain a product A solution; add a mixed solution of solvent B and trifluoroacetic acid to the product A solution, stir and react for 20 - 40 min, perform evaporation and concentration on the reaction solution after the reaction to obtain a solid product, reprecipitate the solid product with anhydrous ether, centrifuge to collect the precipitate, and perform vacuum drying on the precipitate to obtain product B;

[0013] (3) Dissolve PLGA-COOH, EDC, and NHS in solvent B respectively to obtain a PLGA-COOH solution, an EDC solution, and an NHS solution; drop the EDC solution into the PLGA-COOH solution, stir and react for 6 - 20 min; then drop the NHS solution into the PLGA-COOH solution, stir and react for 50 - 70 min, perform reprecipitation on the reaction solution with a methanol-ether mixed solution after the reaction, centrifuge to collect the precipitate, and perform vacuum drying on the precipitate to obtain product C;

[0014] (4) Add product B and product C to solvent B, stir and react for 10 - 14 h. After the reaction is completed, precipitate the reaction solution with anhydrous ether, centrifuge to collect the precipitate after precipitation, and dry the precipitate under vacuum to obtain PEG-PLGA with the hydrophilic segment modified by alendronate.

[0015] According to the tooth-targeted nano-drug-loaded micelle described above, preferably, in step (1), the mass ratio of t-BOC-PEG-NHS to alendronate is (7 - 8):1; more preferably, the mass ratio of t-BOC-PEG-NHS to alendronate is 7.3:1.

[0016] According to the tooth-targeted nano-drug-loaded micelle described above, preferably, the alendronate is sodium alendronate (ALN).

[0017] According to the tooth-targeted nano-drug-loaded micelle described above, preferably, in step (2), the volume ratio of solvent B to trifluoroacetic acid in the mixed solution is 3:1.

[0018] According to the tooth-targeted nano-drug-loaded micelle described above, preferably, in step (3), the mass ratio of PLGA-COOH, EDC and NHS is 100:(8.5 - 10.5):(5 - 7); more preferably, the mass ratio of PLGA-COOH, EDC and NHS is 100:9.58:5.76.

[0019] According to the tooth-targeted nano-drug-loaded micelle described above, preferably, in step (3), the volume ratio of methanol to ether in the methanol-ether mixed solution is 1:1.

[0020] According to the tooth-targeted nano-drug-loaded micelle described above, preferably, in step (4), the mass ratio of product B to product C is 1:1.

[0021] According to the tooth-targeted nano-drug-loaded micelle described above, preferably, according to the tooth-targeted nano-micelle described above, preferably, the hydrophobic antibacterial agent is hydrophobic chlorhexidine.

[0022] According to the tooth-targeted nano-drug-loaded micelle described above, preferably, the preparation method of the hydrophobic chlorhexidine is: dissolve chlorhexidine hydrochloride in solvent C to obtain a chlorhexidine solution; add triethylamine to the chlorhexidine solution, stir and react to obtain hydrophobic chlorhexidine. More preferably, the dosage of triethylamine is 25 L of triethylamine added per 1 mg of chlorhexidine; the stirring reaction time is 3 h.

[0023] According to the tooth-targeted nano-drug-loaded micelle described above, preferably, the solvent A is DMSO, the solvent B is dichloromethane, and the solvent C is DMSO.

[0024] According to the above-mentioned tooth-targeted nano drug-loaded micelles, preferably, the particle size of the tooth-targeted nano micelles ≤ 200 nm.

[0025] The second aspect of the present invention provides a preparation method of the tooth-targeted nano drug-loaded micelles described in the first aspect above, including: adding an amphiphilic block copolymer modified with alendronate to solvent D and stirring evenly, then adding hydrophobic chlorhexidine, and mixing evenly by ultrasonic treatment to obtain a drug-loaded micelle solution; performing dialysis treatment on the drug-loaded micelle solution, and freeze-drying the drug-loaded micelle solution after dialysis to obtain a freeze-dried powder of tooth-targeted nano drug-loaded micelles; wherein, the dialysis solution used for the dialysis treatment is water, and the cut-off molecular weight of the dialysis treatment is 7000 Da.

[0026] According to the above preparation method, preferably, the solvent D is DMSO.

[0027] The third aspect of the present invention provides the application of the tooth-targeted nano drug-loaded micelles described in the first aspect above in the preparation of drugs for preventing, alleviating and treating dental caries.

[0028] The fourth aspect of the present invention provides a drug, which contains the tooth-targeted nano drug-loaded micelles described in the first aspect above and pharmaceutically acceptable excipients.

[0029] Compared with the prior art, the positive and beneficial effects obtained by the present invention are as follows:

[0030] (1) In the present invention, alendronate is connected to the PEG segment of PEG-PLGA to obtain a modified hydrophilic segment PEG, and a PEG-PLGA with a hydrophilic segment modified with alendronate is obtained. The hydrophobic antibacterial agent (such as chlorhexidine) is encapsulated with a PEG-PLGA with a hydrophilic segment modified with alendronate to prepare tooth-targeted nano drug-loaded micelles. The drug-loaded micelles have good antibacterial effects, HA adsorption performance and adsorption effect on human wisdom teeth, which is beneficial to the selective delivery and accumulation of drugs in bone tissue, realizes targeted drug delivery to teeth, and thus better exerts the antibacterial effect of the antibacterial agent on the bacteria causing dental caries, which is of great significance for the treatment and prevention of dental caries.

[0031] (2) The tooth-targeted nano drug-loaded micelles of the present invention show stable drug release performance in both pH 5.0 and pH 7.4 environments, and have better drug release stability in the pH 5.0 environment. The inflammatory site is often acidic. Therefore, the drug-loaded micelles of the present invention will exhibit good drug release performance at the inflammatory lesion site.

[0032] (3) The tooth-targeted nano drug-loaded micelles of the present invention have good safety and in vitro compatibility under different pH conditions. Description of the Drawings

[0033] Figure 1 ALN-PEG prepared for Example 12k -PLGA 20k 1H nuclear magnetic resonance spectrum ( 1 1H-NMR);

[0034] Figure 2 ALN-PEG 2k -PLGA prepared in Example 1 20k Results of DLS particle size distribution of micelles;

[0035] Figure 3 ALN-PEG 2k -PLGA prepared in Example 1 20k TEM (transmission electron microscopy) test results of micelles;

[0036] Figure 4 ALN-PEG 2k -PLGA prepared in Example 1 20k CMC test results;

[0037] Figure 5 Adsorption performance test results of different concentrations of ALN-PEG 2k -PLGA 20k ; where mPEG-PLGA represents mPEG 2k -PLGA 20k and ALN-PEG-PLGA represents ALN-PEG 2k -PLGA 20k ;

[0038] Figure 6 Fluorescence microscopy test photos of the adsorption performance of ALN-PEG-PLGA micelles on teeth; where 100% represents the ALN-PEG 2k -PLGA 20k micelles prepared in Example 1, 50% represents the ALN-PEG 2k -PLGA 20k micelles prepared in Example 4, and 0% represents mPEG 2k -PLGA 20k micelles;

[0039] Figure 7 Fluorescence intensity statistical results of the adsorption performance of ALN-PEG-PLGA micelles on teeth; where 100% represents the ALN-PEG 2k -PLGA 20k micelles prepared in Example 1, 50% represents the ALN-PEG 2k -PLGA 20k micelles prepared in Example 4, and 0% represents mPEG 2k -PLGA 20k micelles;

[0040] Figure 8 is Chlo@ALN-PEG 2k -PLGA 20k Drug release curves of micelles at different pH values and different times;

[0041] Figure 9 is Chlo@ALN-PEG 2k -PLGA 20k Detection results of the biotoxicity of micelles against HGE cells at different pH values; among them, A is the detection result of the biotoxicity of Chlo@ALN-PEG 2k -PLGA 20k micelles against HGE cells; B is the detection result of the biotoxicity of Chlo@ALN-PEG 2k -PLGA 20k micelles against HGE cells;

[0042] Figure 10 is Chlo@ALN-PEG 2k -PLGA 20k Detection results of the antibacterial properties of micelles against Streptococcus mutans at different pH values; among them, A is the detection result of the antibacterial properties of Chlo@ALN-PEG 2k -PLGA 20k micelles against Streptococcus mutans; B is the detection result of the antibacterial properties of Chlo@ALN-PEG 2k -PLGA 20k micelles against Streptococcus mutans; free Chlo. represents chlorhexidine without encapsulation. Specific embodiments

[0043] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. The following embodiments facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all obtained from regular biochemical reagent stores unless otherwise specified. In the following embodiments, quantitative tests are all set with three repeated experiments, and the results are averaged.

[0044] Example 1: Synthesis of ALN-PEG 2k -PLGA 20k (PEG modified with alendronate as the hydrophilic segment 2k -PLGA 20k )

[0045] ALN-PEG 2k -PLGA20k The specific steps of the preparation method are as follows:

[0046] (1) Dissolve 119.1 mg of PEG with t-BOC protecting group and NHS functional group (t-BOC-PEG-NHS, the molecular weight of PEG is 2000 Da) in 4 mL of DMSO to obtain a t-BOC-PEG-NHS solution; dissolve 16.3 g of sodium alendronate (ALN) in ultrapure water to obtain a sodium alendronate solution, wherein the mass ratio of t-BOC-PEG-NHS to ALN is 7.3:1; drop the t-BOC-PEG-NHS solution into the sodium alendronate solution, stir and react for 6 h after dropping, and after the reaction is completed, put the reaction solution into a dialysis bag for dialysis treatment (the dialysis solution used for dialysis treatment is water, and the cut-off molecular weight MWCO of the dialysis bag = 1000 Da) for 3 days, and freeze-dry the dialyzed reaction solution to obtain product A;

[0047] (2) Take 50 mg of product A and put it into a 50 mL two-necked round-bottom flask. After evacuating the two-necked round-bottom flask, fill it with N2 and ventilate for 5 minutes. Then, under the N2 atmosphere, add anhydrous dichloromethane to the two-necked round-bottom flask to fully dissolve product A to obtain a product A solution; drop a mixed solution of 4 - 8 mL of anhydrous dichloromethane and trifluoroacetic acid (the volume ratio of anhydrous dichloromethane to trifluoroacetic acid in the mixed solution is 3:1) into the product A solution, stir and react at room temperature for 0.5 h to remove the protection of the tert-butoxycarbonyl group. After the reaction is completed, concentrate the reaction solution at room temperature using a RE-3000A rotary evaporator to obtain a solid product. The solid product is reprecipitated with 20 times the volume of anhydrous ether, and the precipitate is collected by centrifugation. The precipitate is dried under vacuum to obtain product B;

[0048] (3) Dissolve 100 mg of PLGA-COOH (molecular weight is 20000 Da), 9.58 mg of EDC, and 5.76 mg of NHS respectively with anhydrous dichloromethane to obtain a PLGA-COOH solution, an EDC solution, and an NHS solution; drop the EDC solution into the PLGA-COOH solution, stir and react for 10 min after dropping; then drop the NHS solution into the PLGA-COOH solution, continue to stir and react for 60 min after dropping. After the reaction is completed, reprecipitate the reaction solution with a methanol-ether mixed solution (the volume ratio of methanol to ether in the mixed solution is 1:1), collect the precipitate by centrifugation after reprecipitation, and dry the precipitate under vacuum to obtain product C;

[0049] (4) Add product B (50 mg) and product C (50 mg) to 6 mL of anhydrous dichloromethane, stir and react at room temperature for 12 h. After the reaction, precipitate the reaction solution with 20 times the volume of anhydrous ether. After precipitation, centrifuge to collect the precipitate, and dry the precipitate under vacuum to obtain ALN-PEG 2k -PLGA 20k (i.e., PEG modified with alendronate as the hydrophilic segment 2k -PLGA 20k ).

[0050] Example 2: Synthesis of ALN-PEG 2k -PLGA 10k The synthesis of ALN-PEG

[0051] ALN-PEG 2k -PLGA 10k is basically the same as that in Example 1, except that: in step (3), the molecular weight of PLGA-COOH is 10,000 Da.

[0052] Example 3: Synthesis of ALN-PEG 2k -PLGA 30k The synthesis of ALN-PEG

[0053] ALN-PEG 2k -PLGA 30k is basically the same as that in Example 1, except that: in step (3), the molecular weight of PLGA-COOH is 30,000 Da.

[0054] Example 4: Synthesis of ALN-PEG 2k -PLGA 20k The content of Example 4 is basically the same as that in Example 1, except that: in step (1), 8.15 g of sodium alendronate (ALN) is dissolved in ultrapure water to obtain a sodium alendronate solution, that is, the mass ratio of t-BOC-PEG-NHS to ALN is 14.6:1.

[0055] Example 5: Preparation of tooth-targeted nano-drug-loaded micelles (Chlo@ALN-PEG

[0056] -PLGA 2k -PLGA 20k )

[0057] The preparation method of tooth-targeted nano-drug-loaded micelles (Chlo@ALN-PEG 2k -PLGA 20k ) is specifically as follows:

[0058] S1: Dissolve 2 mg of chlorhexidine hydrochloride in 500 μL of DMSO to obtain a chlorhexidine solution; add 50 μL of triethylamine to the chlorhexidine solution and stir at room temperature for 3 h to obtain hydrophobic chlorhexidine.

[0059] S2: Add the ALN-PEG 2k -PLGA 20k prepared in Example 1 to DMSO, stir and mix evenly, then add the hydrophobic chlorhexidine prepared in step S1, and mix evenly by ultrasonic wave (working frequency 70 mHz, working for 3 s, stopping for 3 s) to obtain a drug-loaded micelle solution; add the drug-loaded micelle solution to a dialysis bag (the cut-off molecular weight MWCO of the dialysis bag = 7000 Da) for dialysis treatment (the dialysis solution used for dialysis treatment is water) for 3 days, and the drug-loaded micelle solution after dialysis is freeze-dried to obtain a tooth-targeted nano drug-loaded micelle lyophilized powder (denoted as Chlo@ALN-PEG 2k -PLGA 20k ).

[0060] Example 6: Preparation of tooth-targeted nano drug-loaded micelles (Chlo@ALN-PEG 2k -PLGA 10k )

[0061] The preparation method of the tooth-targeted nano drug-loaded micelles (Chlo@ALN-PEG 2k -PLGA 10k ) is basically the same as that of the tooth-targeted nano drug-loaded micelles (Chlo@ALN-PEG 2k -PLGA 20k ) in Example 5, and the difference is that in step S2, add the ALN-PEG 2k -PLGA 10k prepared in Example 2 to DMSO, stir and mix evenly.

[0062] Example 7: Preparation of tooth-targeted nano drug-loaded micelles (Chlo@ALN-PEG 2k -PLGA 30k )

[0063] The preparation method of the tooth-targeted nano drug-loaded micelles (Chlo@ALN-PEG 2k -PLGA 30k ) is basically the same as that of the tooth-targeted nano drug-loaded micelles (Chlo@ALN-PEG 2k -PLGA 20k ) in Example 5, and the difference is that in step S2, add the ALN-PEG 2k -PLGA 30k prepared in Example 3 to DMSO, stir and mix evenly.

[0064] Example 8: Preparation of Tooth-Targeted Nano-Drug-Loaded Micelles (Chlo@ALN-PEG 2k -PLGA 20k )

[0065] The content of Example 8 is basically the same as that of Example 5, except that: in step S2, ALN-PEG prepared in Example 4 is added to DMSO 2k -PLGA 20k , and stirred and mixed evenly.

[0066] (I) Characterization and Performance Determination of ALN-PEG-PLGA and Chlo@ALN-PEG 2k -PLGA:

[0067] 1. 1H-NMR Characterization of ALN-PEG 2k -PLGA 20k : 1 The ALN-PEG

[0068] prepared in Example 1 was characterized by 1 1H-NMR, and the characterization results are as 2k shown in 20k Figure 1 .

[0069] Figure 1 As can be seen from, the solvent peak of DMSO-d6 is at 2.485 ppm, the hydrogen characteristic peak of the -CH3 at the end of PLGA is at 1.45 ppm, the hydrogen characteristic peak of the -O-CH2-CO (glycolide) fragment in PLGA is at 4.890 ppm, the hydrogen characteristic peak of the -OCH(CH3)-CO (lactide) fragment in PLGA is at 5.174 ppm, the hydrogen characteristic peak of the -OCH2CH2- fragment of the PEG repeating unit is at 3.495 ppm, and the hydrogen characteristic peak of the -CH2CONHCH2CH2CH2- fragment in ALN is at 2.131 ppm. Through these characteristic peaks, it can be determined that this substance is ALN-PEG 2k -PLGA 20k . Further, by calculating the integral area of the characteristic peaks, the grafting rate of ALN-PEG 2k -PLGA 20k is finally obtained as 59.5%.

[0070]

[0071] 2. Detection of Particle Size, ζ Potential and PdI of ALN-PEG-PLGA and Chlo@ALN-PEG-PLGA​The particle size, ζ potential and PdI of ALN-PEG-PLGA prepared in Examples 1-3 and Chlo@ALN-PEG-PLGA prepared in Examples 5-7 were detected using a Malvern nanoparticle size analyzer (DLS), and the detection results are shown in Table 1 and Figure 2 as follows.

[0072] Table 1 Detection results of particle size, ζ potential and PdI of ALN-PEG-PLGA, Chlo@ALN-PEG 2k -PLGA

[0073] Example Sample Particle size (nm) PdI ζ potential (mV) Example 2 <![CDATA[ALN-PEG 2k -PLGA 10k > 183.44±1.09 0.22±0.07 -0.35±0.12 Example 6 <![CDATA[Chlo@ALN-PEG 2k -PLGA 10k > 245.00±2.15 0.19±0.01 -0.30±0.09 Example 1 <![CDATA[ALN-PEG 2k -PLGA 20k > 38.11±2.03 0.13±0.06 -1.11±0.13 Example 5 <![CDATA[Chlo@ALN-PEG 2k -PLGA 20k > 86.28±1.92 0.10±0.04 -1.93±0.15 Example 3 <![CDATA[ALN-PEG 2k -PLGA 30k > 132.04±1.50 0.33±0.02 -0.11±0.17 Example 7 <![CDATA[Chlo@ALN-PEG 2k -PLGA 30k > 148.70±1.97 0.36±0.05 -0.55±0.21

[0074] As can be seen from Table 1, the micelles of ALN-PEG-PLGA prepared in Examples 1-3 and Chlo@ALN-PEG 2k -PLGA are all negatively charged, indicating that the micelles can be stably distributed in aqueous solution; in addition, the ALN-PEG-PLGA prepared in Examples 2 and 3 and the Chlo@ALN-PEG-PLGA prepared in Examples 6 and 7 are prone to aggregation due to inappropriate hydrophilic-hydrophobic ratios, and the formed micelles have larger and non-uniform particle sizes and larger PdI values; as Figure 2 can be seen, the particle size of the ALN-PEG 2k -PLGA 20k prepared in Example 1 is between 30-50 nm; therefore, the ALN-PEG 2k -PLGA 20k prepared in Example 1 and the micelles prepared in Example 5 have appropriate particle sizes and moderate PdI values. Subsequently, the ALN-PEG-PLGA 20k prepared in Example 1 and the Chlo@ALN-PEG 2k -PLGA 20k prepared in Example 5 were selected as the main research objects.

[0075] The ALN-PEG 2k -PLGA 20k prepared in Example 1 was detected by TEM (transmission electron microscopy), and the detection results are as Figure 3 follows.

[0076] As Figure 3 can be seen, the particle size of the ALN-PEG 2k -PLGA 20k prepared in Example 1 is within 50 nm and is uniform in size. Since the transmission electron microscopy measures the true particle size and the Malvern nanoparticle size analyzer measures the hydrated particle size, there will be some differences between the particle sizes measured by TEM and the nanoparticle size analyzer. However, the particle size of ALN-PEG measured by TEM 2k-PLGA 20k The particle size of the micelles is basically consistent with the particle size detection results of DLS in Table 1.

[0077] 4. Detection of the critical micelle concentration (CMC) of ALN-PEG-PLGA micelles:

[0078] Stability is a fundamental condition for evaluating whether micelles can be used as a drug delivery system. The critical micelle concentration (CMC) is a basic parameter for evaluating the thermodynamic stability of micelles. Micelles are a dynamic structure. When the polymer concentration is higher than the CMC, the polymer can self-assemble to form micelles; while when the polymer concentration is lower than the CMC, the micelles will disassemble and return to the single molecular chain state. In the present invention, the CMC of ALN-PEG 2k -PLGA 20k prepared in Example 1 was determined by the pyrene fluorescence probe method.

[0079] The specific method steps are as follows: Take 10 μL of an acetone solution with a pyrene concentration of 1.78×10 -4 mol / mL and add it to a 10 mL round-bottom flask. Evaporate the acetone under vacuum, and then add 3 mL of polymer (ALN-PEG 2k -PLGA 20k ) solutions with different concentrations (in the range of 0.02 - 1000 μg / mL) to the round-bottom flask containing pyrene residues. The final concentration of pyrene in the polymer solution is 6×10 -7 mol / mL, and equilibrate for 24 hours at room temperature. Use a F-280 fluorescence spectrophotometer from Tianjin Gangdong Company. The excitation wavelength is 335 nm, the excitation and emission slit widths are 5 nm, and measure the emission spectrum of the polymer in the range of 350 - 440 nm. By measuring the fluorescence intensities of the characteristic peaks of pyrene at 373 nm and 393 nm, namely I1 and I3, at a series of different polymer concentrations, plot with the concentration on the X-axis and I3 / I1 on the Y-axis, make a horizontal tangent to the curve and a tangent to the deformed curve, and the concentration value corresponding to the intersection point is the CMC value of the polymer.

[0080] The detection results of CMC are as Figure 4 shown. As Figure 4 can be seen, the CMC value of ALN-PEG 2k -PLGA 20k prepared in Example 1 is 2.29 μg / mL, indicating that the hydrophobic segment PLGA of ALN-PEG-PLGA 20k is relatively long, the hydrophobic core is relatively tight, and the stability is good.

[0081] 5. Study on the adsorption performance of ALN-PEG-PLGA 20k on hydroxyapatite (HA)

[0082] Taking the ALN-PEG 2k -PLGA 20k prepared in Example 1 as an example, the adsorption performance of the ALN-PEG 2k -PLGA 20k micelles on hydroxyapatite (HA) was studied. At the same time, mPEG 2k -PLGA 20k micelles were used as a comparison.

[0083] The specific experimental method was as follows: The ALN-PEG 2k -PLGA 20k micelles were formulated into different ratios (equal masses of ALN-PEG 2k -PLGA 20k micelles and mPEG 2k -PLGA 20k micelles were respectively dissolved in the same volume of water to obtain ALN-PEG 2k -PLGA 20k micelle solution and mPEG 2k -PLGA 20k micelle solution; The ALN-PEG 2k -PLGA 20k micelle solution and mPEG 2k -PLGA 20k micelle solution were mixed at a volume ratio of 2:8 to obtain 20% ALN-PEG 2k -PLGA 20k ; The ALN-PEG 2k -PLGA 20k micelle solution and mPEG 2k -PLGA 20k micelle solution were mixed at a volume ratio of 4:6 to obtain 40% ALN-PEG 2k -PLGA 20k ; The ALN-PEG 2k -PLGA 20k micelle solution and mPEG 2k -PLGA 20k micelle solution were mixed at a volume ratio of 8:2 to obtain 80% ALN-PEG 2k -PLGA 20k; ) After mixing with the HA suspension, stir and adsorb for 30 minutes and 24 hours respectively, then centrifuge to obtain the supernatant. Rinse the lower-layer solid with ionized water three times to wash away the micelles that have not been successfully adsorbed, obtaining the washed-out liquid. Then, lyophilize the supernatant and the washed-out liquid together. Dissolve the lyophilized powder in DMSO and measure the ultraviolet fluorescence absorption value at a wavelength of 253 nm. As a result, the amount of micelles that have not been successfully adsorbed on HA is obtained, and it is compared with the ultraviolet fluorescence absorption value of the drug micelles with the same concentration that have not undergone the HA adsorption test at a wavelength of 253 nm, thereby indirectly reflecting the adsorption effect of the micelles on HA. The experimental results are as Figure 5 shown.

[0084] As Figure 5 can be seen, as the proportion of ALN-PEG 2k -PLGA 20k increases, the absorbance values of the supernatant and the washed-out liquid gradually decrease, indirectly indicating that the amount of targeted drug-loaded micelles adsorbed on HA gradually increases, and its increase is directly related to the increase in the proportion of ALN-PEG 2k -PLGA 20k . At the same time, by comparing the stirring adsorption for 30 minutes with the stirring adsorption for 24 hours, it can be found that the overall adsorption effect of the stirring adsorption for 24 hours is better than that for 30 minutes. Although the effect is not very obvious, it is speculated that the possible reason is that sodium alendronate itself has good adsorption to hydroxyapatite, and in addition, the adsorption object is a HA turbid liquid with a large specific surface area, so a high proportion of adsorption can be achieved in a short time. Therefore, the amount adsorbed after 24 hours does not increase much. From this, it can be preliminarily known that the ALN-PEG 2k -PLGA 20k micelles can achieve selective drug delivery and accumulation in bone tissue, reflecting the value of its application in dental targeted nano-drug delivery formulations.

[0085] 6. Study on the adsorption performance of ALN-PEG-PLGA micelles on teeth

[0086] The method of loading coumarin 6 (C6) fluorescence was used to evaluate the adsorption performance of the ALN-PEG 2k -PLGA 20k micelles prepared in Example 1 and Example 4 on human wisdom teeth, and at the same time, mPEG 2k -PLGA 20k micelles were used as a comparison.

[0087] The specific experimental method is as follows:

[0088] 10 mg of ALN-PEG 2k -PLGA 20k1 mg of the C6 solution was dissolved in 500 μL of DMSO, and then the two solutions prepared above were mixed evenly to obtain a mixed solution. The mixed solution was added dropwise into 9 mL of deionized water under an ultrasonic environment with a working frequency of 70 mHz, working for 3 s and stopping for 3 s, and ultrasonic treatment was carried out for 5 minutes. Then the obtained solution was loaded into a dialysis bag (MWCO = 3500 Da) and dialyzed with deionized water as the dialysis solution for two days. Then, 2 mL of the dialyzed mixed solution was taken into a 5 mL centrifuge tube, wrapped with tin foil for light shielding treatment, and six healthy human wisdom teeth of uniform size that had been polished beforehand were placed into the centrifuge tube and soaked for 3 minutes and 24 hours respectively. After the soaking was completed, they were washed three times in PBS buffer for 1 minute each time and then placed under an inverted fluorescence microscope for observation, and the fluorescence intensity of the fluorescence images taken by the fluorescence microscope was measured using Image J software. The test results are as Figure 6 and Figure 7 shown.

[0089] It can be seen from Figure 6 and Figure 7 that under the same soaking time, the fluorescence image field of view with a higher ALN-PEG 2k -PLGA 20k micelle ratio is brighter and the fluorescence intensity is higher, indicating that more micelles are adsorbed on the wisdom teeth; under the same micelle doping ratio, the general view of the 24-hour soaking is brighter and the fluorescence intensity is lower than that of the 3-minute soaking, indicating that with the increase of the soaking time, more micelles are adsorbed on the tooth surface.

[0090] (II) Performance detection of Chlo@ALN-PEG-PLGA

[0091] 1. Detection of drug loading and encapsulation efficiency of Chlo@ALN-PEG-PLGA

[0092] Taking the Chlo@ALN-PEG 2k -PLGA 20k prepared in Example 5 as an example, the yield, drug loading and encapsulation efficiency of Chlo@ALN-PEG-PLGA micelles were detected. The calculation formulas for drug loading and encapsulation efficiency are as follows: Yield% = feeding mass / product mass × 100; Drug loading% = mass of encapsulated drug / (mass of input drug + mass of carrier) × 100; Drug loading% = mass of encapsulated drug / mass of input drug × 100;

[0093] After detection, the yield of the drug-loaded micelles of Chlo@ALN-PEG 2k -PLGA 20k prepared in Example 5 was 81.8%, the encapsulation efficiency was 67.8%, and the drug loading was 5.2%.

[0094] 2. In vitro drug release study of Chlo@ALN-PEG-PLGA

[0095] Taking Chlo@ALN-PEG 2k -PLGA prepared in Example 5 20k as an example, the in vitro drug release study of Chlo@ALN-PEG-PLGA micelles was carried out.

[0096] The specific experimental method was as follows: At 37 °C, the Chlo@ALN-PEG 2k -PLGA 20k micelles prepared in Example 5 were respectively added to PBS buffer solution (pH = 7.4) and disodium hydrogen phosphate-citrate buffer solution (pH = 5.0), and the cumulative release amounts of the drug (chlorhexidine) from the Chlo@ALN-PEG 2k -PLGA 20k micelles at different pH values and different times were detected. The experimental results are as Figure 8 shown.

[0097] As can be seen from Figure 8 the results, at the initial stage of drug release, there was an initial burst release phenomenon for the Chlo@ALN-PEG 2k -PLGA 20k micelles in both PBS buffer solution (pH = 7.4) and disodium hydrogen phosphate-citrate buffer solution (pH = 5.0). In the range of 0 - 7 hours, the release amounts of the Chlo@ALN-PEG 2k -PLGA 20k micelles in the two solutions were relatively obvious. At 7 hours, the cumulative release amount of the Chlo@ALN-PEG 2k -PLGA 20k micelles in PBS buffer solution reached 58%, while the cumulative release amount in disodium hydrogen phosphate-citrate buffer solution reached 71%. After 7 hours, the drug release amounts at each time point slowly decreased, and the cumulative release amounts gradually tended to a plateau. At 100 hours, the cumulative release amount of the Chlo@ALN-PEG 2k -PLGA 20k micelles in PBS buffer solution reached 75%, and the cumulative release amount in disodium hydrogen phosphate-citrate buffer solution could reach 90%. In addition, the drug release effect of the Chlo@ALN-PEG 2k -PLGA 20k micelles in disodium hydrogen phosphate-citrate buffer solution was better than that in PBS buffer solution, and at each point during the drug release process, each set of parallel samples was relatively uniform. It can be inferred from this that Chlo@ALN-PEG 2k -PLGA 20kMicelles are more conducive to the release of chlorhexidine in an acidic environment; the inflammatory site is often acidic, so the drug-loaded micelles will exhibit good drug release performance at the inflammatory lesion site.

[0098] 3. Cytotoxicity study of Chlo@ALN-PEG-PLGA on HGE cells (human gingival epithelial cells)

[0099] Taking Chlo@ALN-PEG prepared in Example 5 2k -PLGA 20k as an example, the MTT method was used to study the cytotoxicity of Chlo@ALN-PEG-PLGA micelles on HGE cells (human gingival epithelial cells) under different pH conditions (pH 7.4 and pH 5.0). At the same time, ALN-PEG-PLGA prepared in Example 1 20k was used as a control.

[0100] The specific experimental method was as follows: Human gingival epithelial cells (HGE) were inoculated into 96-well plates at a concentration of 5000 per well and cultured overnight in a constant temperature incubator at 37°C and a CO2 concentration of 5% to allow the cells to adhere. Samples were added. The samples were set at 9 concentrations of Chlo@ALN-PEG 2k -PLGA 20k micelles at 25, 50, 100, 200, 300, 400, 500, 800, and 1000 μg / mL respectively. Each concentration had 4 replicate parallel samples. At the same time, DMEM medium was used as a blank control, and the cells were co-incubated at 5% CO2 and 37°C for 24 hours. Then, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution with a concentration of 5 mg / mL was added to each well. After incubation for 4 hours, the medium was aspirated, 150 μL of DMSO was added, and it was placed on a shaker and shaken at low speed for 10 minutes to fully dissolve the crystals. The absorbance value of each well was measured at a wavelength of 570 nm using an enzyme-linked immunosorbent assay detector, and the cell survival rate was calculated based on the absorbance value. The cell survival rate % = mean absorbance value of the sample group / mean absorbance value of the blank control group × 100. The experimental results are as Figure 9 shown.

[0101] As Figure 9 can be seen, at pH 7.4, when the concentration of Chlo@ALN-PEG 2k -PLGA 20k micelles was 1000 μg / mL, the cell survival rate was still higher than 80%; at pH 5.0, when the concentration of Chlo@ALN-PEG 2k -PLGA 20k micelles did not exceed 800 μg / mL, the cell survival rate remained above 80%. This shows that Chlo@ALN-PEG of the present invention2k -PLGA 20k Micelles, ALN-PEG 2k -PLGA 20k The micelles have good safety at different pH values, low cytotoxicity, and good in vitro compatibility.

[0102] 4. Antibacterial Performance Study of Chlo@ALN-PEG-PLGA against Streptococcus mutans

[0103] Using Chlo@ALN-PEG prepared in Example 5 2k -PLGA 20k as an example, the antibacterial performance of Chlo@ALN-PEG-PLGA micelles against Streptococcus mutans under different pH conditions (pH 7.4 and pH 5.0) was studied by the minimum inhibitory concentration method, and chlorhexidine without encapsulation was used as a comparison.

[0104] The specific experimental method is as follows: Wash the activated bacteria test tubes, plug the lids but not tightly, wrap them with kraft paper. Seal the conical flasks containing brain heart infusion broth and plate count agar with kraft paper. Wrap the pipette tips and centrifuge tubes of different specifications with kraft paper. Put the above items into a high-temperature steam sterilizer and sterilize at 120 °C for 30 minutes. Take them out after the temperature drops to 60 °C and place them in an oven for later use. Prepare ten bacterial culture plates. Pour the solid medium into the bacterial culture plates while it is hot, about 20 mL per plate. Let it cool, place it in a laminar flow hood overnight, and blow off the water vapor. Take out Streptococcus mutans from the -80 °C refrigerator. Heat the inoculation loop until it is red-hot and then let it cool completely. Dip the culture loop into the culture medium to pick up the bacterial strain and place it in the liquid medium, and then culture it overnight on a shaker at 37 °C. Take the overnight bacteria and the liquid medium and prepare 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 . Take 20 μL of 10 -5 、10 -6 、10 -7 、10 -8 bacterial suspensions and spread them evenly on the solid agar plates. Spread two plates for each concentration, and at the same time spread two blank plates (only the medium). Then count the number of colonies after culturing at 37 °C for 24 hours.

[0105] Take the freeze-dried powder of Chlo@ALN-PEG prepared in Example 5 2k -PLGA 20k micelles, dissolve them with LB liquid broth, and add the bacterial suspension (10 7CFU / mL) to make the chlorhexidine concentrations be 0, 2, 4, 8, 16, 32, 64, and 128 μg / mL respectively. 200 μL of the bacterial suspension containing Chlo@ALN-PEG 2k -PLGA 20k micelles was taken into a 96-well plate, incubated at a constant temperature of 37 °C for 24 hours, and the absorbance was read on an enzyme-linked immunosorbent assay (ELISA) reader; meanwhile, unencapsulated chlorhexidine (i.e., free chlorhexidine) was used as a positive control. Referring to the International Organization for Standardization 22196 (ISO 22196) standard, the bacterial survival rate was calculated, and the formula for calculating the bacterial survival rate was: Bacterial survival rate % = average absorbance of the sample group / average absorbance of the positive control group × 100. The experimental results are as Figure 10 shown.

[0106] As Figure 10 can be seen, as the concentration of Chlo@ALN-PEG 2k -PLGA 20k micelles increased, the survival rate of Streptococcus mutans gradually decreased; in addition, when comparing Chlo@ALN-PEG 2k -PLGA 20k micelles with the same drug concentration and unencapsulated chlorhexidine, it was found that the antibacterial effect of unencapsulated chlorhexidine was better than that of Chlo@ALN-PEG 2k -PLGA 20k micelles. It was speculated that the possible reason was that aggregation and drug release occurred during the dialysis of Chlo@ALN-PEG 2k -PLGA 20k micelles, and during the subsequent antibacterial experiment, the actual drug loading of the chlorhexidine-loaded micelles was less than the theoretical drug loading; on the other hand, according to the previous drug release performance study, Chlo@ALN-PEG 2k -PLGA 20k micelles did not release all the drugs within 24 hours, only most of the chlorhexidine was released. Therefore, relatively speaking, the amount of drugs released by Chlo@ALN-PEG 2k -PLGA 20k micelles into the external environment for antibacterial was less. Generally speaking, as the concentration of the loaded chlorhexidine increased, the antibacterial effect became better, indicating that the Chlo@ALN-PEG 2k -PLGA 20k micelles prepared in this invention had a good antibacterial effect on Streptococcus mutans and could be used for the prevention, alleviation, and treatment of dental caries.

[0107] The above embodiments are specific implementation manners of the present invention, but the implementation manners of the present invention are not limited to the above embodiments. Any other combinations, changes, modifications, substitutions, and simplifications that do not exceed the design concept of the present invention fall within the protection scope of the present invention.

Claims

1. A tooth-targeted nano-drug-loaded micelle, characterized in that, The tooth-targeting nano-micelles include copolymer micelles and a hydrophobic drug encapsulated by the copolymer micelles. The copolymer is an amphiphilic block copolymer modified with alendronate, and the hydrophobic drug is a hydrophobic antibacterial agent.

2. The tooth-targeted nano-drug-loaded micelle according to claim 1, characterized in that, The copolymer is PEG-PLGA with a hydrophilic segment modified with alendronate.

3. The tooth-targeted nano-drug-loaded micelle according to claim 2, wherein In the PEG-PLGA with a hydrophilic segment modified with alendronate, the molecular weight of the PEG segment is 2000 Da, and the molecular weight of the PLGA segment is 20000 Da.

4. The tooth-targeted nano-drug-loaded micelle according to claim 2, wherein The preparation method of PEG-PLGA with a hydrophilic segment modified with alendronate is as follows: (1) Dissolve t-BOC-PEG-NHS in solvent A to obtain a t-BOC-PEG-NHS solution; drop the t-BOC-PEG-NHS solution into the alendronate solution, stir and react for 5 - 6 h after dropping, perform dialysis treatment on the reaction solution after the reaction, and perform lyophilization on the dialyzed reaction solution to obtain product A; (2) Under an inert gas atmosphere, dissolve the product A in solvent B to obtain a product A solution; add a mixed solution of solvent B and trifluoroacetic acid to the product A solution, stir and react, perform evaporation and concentration on the reaction solution after the reaction to obtain a solid product, re-precipitate the solid product with anhydrous ether and then centrifuge to collect the precipitate, and perform vacuum drying on the precipitate to obtain product B; (3) Dissolve PLGA-COOH, EDC, and NHS in solvent B respectively to obtain a PLGA-COOH solution, an EDC solution, and an NHS solution; Drop the EDC solution into the PLGA-COOH solution, stir and react; then drop the NHS solution into the PLGA-COOH solution, stir and react, perform re-precipitation on the reaction solution with a methanol-ether mixed solution after the reaction, re-precipitate and then centrifuge to collect the precipitate, and perform vacuum drying on the precipitate to obtain product C; (4) Add product B and product C to solvent B, stir and react for 10 - 14 h, perform re-precipitation on the reaction solution with anhydrous ether after the reaction, re-precipitate and then centrifuge to collect the precipitate, and perform vacuum drying on the precipitate to obtain PEG-PLGA with a hydrophilic segment modified with alendronate.

5. The tooth-targeted nano drug-loaded micelle according to any one of claims 1-4, characterized in that, The hydrophobic antibacterial agent is hydrophobic chlorhexidine.

6. The tooth-targeted nano-drug-loaded micelle according to claim 5, characterized in that, The preparation method of the hydrophobic chlorhexidine is as follows: dissolve chlorhexidine hydrochloride in solvent C to obtain a chlorhexidine solution; add triethylamine to the chlorhexidine solution, stir and react to obtain hydrophobic chlorhexidine.

7. The tooth-targeted nano-drug-loaded micelle according to claim 1, characterized in that, The particle size of the tooth-targeting nano-micelles is ≤200 nm.

8. The preparation method of the tooth-targeted nano drug-loaded micelle according to any one of claims 1-7, characterized in that, It includes: Add the amphiphilic block copolymer modified with alendronate to solvent D, stir and mix evenly, then add hydrophobic chlorhexidine, and mix evenly by ultrasonic to obtain a drug-loaded micelle solution; Perform dialysis treatment on the drug-loaded micelle solution, and perform lyophilization on the dialyzed drug-loaded micelle solution to obtain a freeze-dried powder of tooth-targeting nano-drug-loaded micelles; wherein, the dialysis solution used for the dialysis treatment is water, and the cut-off molecular weight of the dialysis treatment is 7000 Da.

9. Use of the tooth-targeting nano-drug-loaded micelles according to any one of claims 1 - 7 in the preparation of a drug for preventing, alleviating, and treating dental caries.

10. A drug, which contains the tooth-targeted nano-drug-loaded micelle according to any one of claims 1-7 and a pharmaceutically acceptable excipient.

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