A dual-pH responsive charge-reversal-mineralization ion release liposome preparation, preparation method and application thereof

Abstract

The application discloses a double pH response charge flip-mineralization ion release liposome preparation and a preparation method and application thereof. An ethanol solution of a mineralization functional nanometer material is mixed with a chloroform solution of 1,2-dioleoyl-sn-glycero-3-phosphatidic acid sodium salt, and then ultrasonic water bath and centrifugal collection of the precipitate are carried out, and the precipitate is resuspended in chloroform; (R)-2,3-bis(palmitoyloxy)propyl(2-(trimethylammonium)ethyl) phosphate, DCPA and DSPE-mPEG 2000 are dissolved in chloroform; after mixing and stirring of the mixture obtained in the above two steps at room temperature, a dioxane solution containing TPAI is added, and chloroform is removed by rotary evaporation to obtain a lipid membrane, and the lipid membrane is subjected to hydration, centrifugation and dialysis to obtain the double pH response charge flip-mineralization ion release liposome preparation. The application can be used for removing caries-causing biofilms and promoting remineralization of demineralized enamel in early caries treatment, and provides a new treatment strategy for caries prevention and treatment.

Description

Technical Field

[0001] This invention relates to the field of liposome materials, and in particular to a dual pH-responsive charge-flipping-mineralization ion-releasing liposome formulation, its preparation method, and its application. Background Technology

[0002] Dental caries is a highly prevalent chronic oral disease worldwide, with a prevalence rate of up to 35% in permanent teeth. The occurrence of dental caries is the result of long-term interactions among multiple factors, including the host, bacteria, food, and time. Among these factors, cariogenic biofilms, represented by Streptococcus mutans, play a central role in the disease process. When external factors disrupt the homeostasis of the local microecology of dental plaque, dominant acid-producing and acid-tolerant bacteria can rapidly utilize fermentable carbohydrates to produce large amounts of organic acids, causing a continuous decrease in local pH. This induces demineralization of tooth tissue and gradually forms caries lesions, ultimately leading to defects in the hard tissues of the tooth. As the lesion progresses deeper, caries can further affect the pulp and periapical tissues, leading to pulpitis and periapical periodontitis, thus seriously impacting the patient's overall health and quality of life. Therefore, effectively removing cariogenic bacteria and their biofilms, regulating and reversing the acidified cariogenic microenvironment, and promoting remineralization of the hard tissues of the tooth are of great significance for the prevention and treatment of dental caries.

[0003] Current caries management strategies primarily focus on inhibiting bacterial activity and biofilm growth to reduce acid production and lower the risk of tooth demineralization. Common clinical approaches include mechanical debridement, fluoride application, and various antibiotic treatments. However, these methods still have significant limitations. For example, mechanical intervention is invasive and inevitably involves additional removal of healthy tooth tissue; while fluoride promotes remineralization, excessive use can lead to adverse reactions such as fluorosis; antibiotics not only have limited penetration into mature cariogenic biofilms, making it difficult to exert effective bactericidal effects within the biofilm, but may also induce drug resistance with long-term use, reducing treatment efficacy. Furthermore, these traditional methods are difficult to effectively reverse existing demineralization lesions and cannot achieve functional regeneration of tooth hard tissues. Therefore, developing novel treatment strategies that target cariogenic biofilms and combine antibacterial and remineralization functions has become a research hotspot in caries prevention and treatment. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a dual pH-responsive charge-flipping-mineralization ion-releasing liposome formulation, its preparation method, and its application.

[0005] In a first aspect, the present invention provides a method for preparing a charge-flipped-mineralized ion-releasing liposome formulation with dual pH response, which is achieved through the following technical solution.

[0006] A method for preparing a dual pH-responsive charge-flipped-mineralized ion-releasing liposome formulation includes the following steps: S1. Preparation of mineralized functional nanomaterials S2. Preparation of dual pH-responsive charge-flipping-mineralized ion-releasing liposome nanoparticles a. Mix an ethanol solution containing mineralized nanomaterials with a chloroform solution of sodium 1,2-dioleoyl-sn-glycerol-3-phosphatidyl acid, sonicate in a water bath, centrifuge to collect the precipitate, and resuspend it in chloroform; wherein the mass ratio of mineralized nanomaterials to sodium 1,2-dioleoyl-sn-glycerol-3-phosphatidyl acid is (1-20):1; b. Add (R)-2,3-bis(palmitoyloxy)propyl(2-(trimethylammonium)ethyl)phosphate, DCPA, and DSPE-mPEG. 2000 Dissolving in chloroform yields a lipid mixture; wherein (R)-2,3-bis(palmitoyloxy)propyl(2-(trimethylammonium)ethyl)phosphate, DCPA, and DSPE-mPEG 2000 The mass ratio is 4 : (1.6-2.4) : (2.0-4.0); c. Mix the mixture obtained in step a with the mixture obtained in step b at room temperature and stir for 12-24 h. Then add a dioxane solution containing TPAI and mix well. Remove chloroform by rotary evaporation to obtain a lipid membrane. Hydrate and centrifuge the lipid membrane to obtain a nanoparticle solution. Finally, dialyze to obtain a dual pH-responsive charge-flipping-mineralized ion-releasing liposome formulation. The mass ratio of mineralized functional nanomaterials, lipid mixture and TPAI is (0.24-0.32): 8.5: (0.18-0.23).

[0007] Furthermore, in step S1, the mineralization functional nanomaterial is an inorganic nanomaterial capable of releasing calcium ions and / or promoting the formation of calcium phosphate mineralization; the mineralization functional nanomaterial is selected from calcium carbonate, calcium phosphate nanomaterials, bioactive glass, calcium peroxide and combinations thereof.

[0008] Furthermore, the preparation method of calcium carbonate nanoparticles is as follows: anhydrous calcium chloride is dissolved in water and diluted with anhydrous ethanol. The resulting mixture and ammonium bicarbonate are placed in two separate containers and reacted together at 35-45℃ for 6-12 hours. The reaction product is centrifuged to obtain calcium carbonate nanoparticles. The mass ratio of calcium chloride to ammonium bicarbonate is (1-3):100.

[0009] Furthermore, in step S2a, the ultrasonic water bath time is 20-30 min; the centrifugation conditions are: centrifugation at 8000-10000 rpm for 10-15 min.

[0010] Furthermore, in step S2b, DSPE-mPEG 2000 Its molecular weight is 2000.

[0011] Furthermore, in step S2c, the lipid membrane hydration treatment method is as follows: place the lipid membrane in water and ultrasonically bathe it at 45-50℃ for 1-3 minutes.

[0012] Secondly, the present invention provides a dual pH-responsive charge-flipped-mineralized ion-releasing liposome formulation, which is achieved through the following technical solution.

[0013] A dual pH-responsive charge-flipped-mineralized ion-releasing liposome formulation prepared by the above preparation method.

[0014] Thirdly, the present invention provides the use of a dual pH-responsive charge-flipped-mineralized ion-releasing liposome formulation, which is achieved through the following technical solution.

[0015] Application of a charge-flipping-mineralization ion-releasing liposome formulation with dual pH response in the preparation of products for treating dental caries.

[0016] This application has the following beneficial effects: This invention's dual pH-responsive charge-flipping-mineralization ion-releasing liposome formulation can effectively penetrate and disrupt biofilm structures, eliminating cariogenic bacteria, and the released mineralization ions can promote enamel repair and remineralization. This invention can be used for the early treatment of caries by clearing cariogenic biofilms and promoting the remineralization of demineralized enamel, providing a new treatment strategy for caries prevention and treatment. Attached Figure Description

[0017] Figure 1 This is a transmission electron microscope image of the nanoparticles prepared in this invention; Figure 2 This is the ultraviolet-visible spectrum of the nanoparticles prepared by this invention; Figure 3 This is a Zeta potential diagram of the nanoparticles prepared by this invention; Figure 4 This is a graph showing the photothermal properties of the nanoparticles prepared according to the present invention; Figure 5 This is a graph showing the photodynamic properties of the nanoparticles prepared in this invention; Figure 6 This is a graph showing the calcium release results of the nanoparticles in Example 1 of the present invention; Figure 7 This is a graph showing the results of the nanoparticles in Example 1 of this invention inhibiting bacterial acid production; Figure 8 This is a graph showing the permeation experiment results of the nanoparticle biomembrane prepared by this invention; Figure 9 This is a diagram showing the bactericidal effect of the nanoparticles prepared in this invention on the biofilm of Streptococcus mutans under light and non-light conditions. Figure 10 This is a diagram showing the effect of the nanoparticles prepared in this invention on the removal of Streptococcus mutans biofilm. Detailed Implementation

[0018] The invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise specified, the experimental methods used in the embodiments of the present invention are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0019] The sodium 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPA) used in the following embodiments of the present invention was purchased from Aladdin D130369-100mg.

[0020] The (R)-2,3-bis(palmitoyloxy)propyl(2-(trimethylammonium)ethyl)phosphate (DPPC) used in the following embodiments of the present invention was purchased from Bid Pharmaceutical BD53403-1g.

[0021] The preparation method of 2-(4-((1,5-bis(octadecoxy)-1,5-dioxopentan-2-yl)carbamoyl)pyridin-1-onthiol-1-yl)acetate (DCPA) used in the following embodiments of the present invention can be found in: R. Li, B. Liu, C. Wang, Z. Sun, S. Lv, L. Tian, ​​M. Xiao, Q. Zheng, L. Shi, C. Zhu, BMEMat2026, 4,e70014.

[0022] The following embodiments of the present invention use distearate-mPEG (distearate-phosphatidylethanolamine-methoxy polyethylene glycol 2000). 2000 Purchased from Bid Pharmaceutical BD01106217-250mg.

[0023] The preparation method of 5-[5-(4-(bis(4-methoxyphenyl)amino)phenyl)-2-thienylmethylene]-3-(dicyanomethylene)-1-indanone (TPAI) used in the following embodiments of the present invention can be found in: Z. Sun, M. Xiao, S. Lv, C. Wang, H. Fu, L. Tian, ​​L. Shi, C. Zhu, A pH-Responsive, Surface Charge-Switchable Nanosystem with Enhanced Biofilm Penetration for Synergistic Photodynamic and Antibiotic Therapy of Diabetic Wounds. Adv. Funct. Mater. 2025, 35, 2418711.

[0024] The dioxane used in the following embodiments of the present invention was purchased from Maclean D807836-500mL.

[0025] Example 1

[0026] A method for preparing a dual pH-responsive charge-flipped-mineralized ion-releasing liposome formulation includes the following steps: (1) Preparation of calcium carbonate nanoparticles Weigh 150 mg of anhydrous calcium chloride and dissolve it in 200 μL of pure water. Dilute it with 100 ml of anhydrous ethanol and place it in a beaker. Seal the beaker with aluminum foil and leave 12 pores with a diameter of about 1 mm. Place the beaker and a glass bottle containing 5 g of ammonium bicarbonate in a vacuum drying oven and react at 40 °C for 6 h. Centrifuge the product in the beaker (8000 rpm for 10 min) to obtain white precipitate of calcium carbonate nanoparticles (CaCO3). Add 10 ml of anhydrous ethanol and disperse it by ultrasonication. Centrifuge at 3000 rpm for 5 min to remove large particles from the solution. Take the supernatant for later use.

[0027] (2) Preparation of CaCO3 / TPAI@Lipo-PyB nanoparticles a. Take 5 ml of CaCO3 ethanol solution (2 mg / ml) prepared in step (1) and 1 ml of 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine sodium salt (DOPA) chloroform solution (2 mg / ml), sonicate in a water bath for 20 min, centrifuge at 8000 rpm for 10 min, collect the precipitate and resuspend it in 2 ml of chloroform; b. 4 mg (R)-2,3-bis(palmitoyloxy)propyl(2-(trimethylammonium)ethyl) phosphate (DPPC), 2 mg DCPA, and 2.5 mg DSPE-mPEG. 2000 Dissolve in 850 μL of chloroform; c. The mixture obtained in step a and the mixture obtained in step b were mixed and stirred at room temperature for 12 h. Then, 40 μL of a 10 mM dioxane solution containing TPAI was added and mixed thoroughly. Chloroform was removed by rotary evaporation, forming a thin and uniform lipid film on the inner wall of the flask. 3 ml of deionized water was added, and the lipid film was hydrated in an ultrasonic water bath at 48 °C. The resulting solution was centrifuged at 3000 rpm for 5 min, and the precipitate was discarded to obtain a stable drug-loaded nanoparticle solution. Finally, after dialyzing for 24 h to remove free calcium ions, CaCO3 / TPAI@Lipo-PyB nanoparticles were obtained and stored at 4 °C for later use.

[0028] Comparative Example 1 The difference from Example 1 is that in step (2)b, only 6 mg DPPC and 2.5 mg DSPE-mPEG are added. 2000 Dissolved in chloroform. The final product was CaCO3 / TPAI@Lipo nanoparticles that did not exhibit pH-responsive charge-flipping properties.

[0029] Comparative Example 2 The difference from Example 1 is that in step (2)c, 40 μL of dioxane solution (10 mM) containing TPAI is not added. CaCO3@Lipo-PyB nanoparticles were finally prepared.

[0030] Comparative Example 3 The difference from Example 1 is that in step (2)b, only 6 mg DPPC and 2.5 mg DSPE-mPEG are added. 2000 Dissolved in chloroform; in step (2)c, 40 μL of dioxane solution (10 mM) containing TPAI was not added. CaCO3@Lipo nanoparticles were finally prepared.

[0031] Performance testing 1. Physicochemical characterization of liposome nanosystems (1) Nanoparticle characterization The particle size of different nanoparticles was tested by dynamic light scattering, and the results are shown in the table below: Table 1. Nanoparticle size and polydispersity index (PDI)

[0032] The above results show that the four types of nanoparticles prepared in this invention are all uniformly dispersed, with an average particle size of approximately 100 nm and a polydispersity index (PDI) of less than 0.3, indicating that the system has good uniformity and stability. Figure 1 As shown, transmission electron microscopy reveals that the prepared nanoparticles exhibit a typical liposome-like spherical vesicle structure with intact morphology.

[0033] (2) UV-Vis spectrum of nanoparticles like Figure 2 As shown, the maximum absorption wavelength of TPAI was measured to be approximately 630 nm, and the prepared nanoparticles also exhibited a significant absorption peak at the same wavelength, indicating that TPAI has been successfully loaded into the nanoparticles.

[0034] (3) Zeta potential of nanoparticles like Figure 3 As shown, the charge-flipping performance of liposomes was verified by measuring the Zeta potential of nanoparticles in buffer solutions with different pH values. The results showed that CaCO3 / TPAI@Lipo-PyB exhibited significant pH-responsive charge-flipping characteristics: its Zeta potential was -2.83 mV at pH 7.4, transitioning to a positive potential (+2.535 mV) at pH 6.5, and gradually increasing to +10.1 mV at lower pH conditions. In contrast, the liposomes CaCO3 / TPAI@Lipo, lacking charge-flipping groups, maintained a negative potential (approximately -7 mV) under different pH conditions, demonstrating no charge-flipping ability.

[0035] (4) Photothermal properties of nanoparticles like Figure 4 As shown, at 0.9 W / cm 2 Under 660 nm laser irradiation, both groups of TPAI-loaded liposome nanoparticle solutions (CaCO3 / TPAI@Lipo-PyB and CaCO3 / TPAI@Lipo, TPAI = 40 μM) exhibited significant photothermal effects: the solution temperature continuously increased from 21 ℃ and eventually stabilized at approximately 48 ℃. In contrast, the other three control samples (Blank, CaCO3@Lipo-PyB, and CaCO3@Lipo) showed only a temperature increase from 21 ℃ to approximately 22 ℃ under the same conditions, with almost no significant change, indicating that the photothermal temperature rise mainly originated from the effective loading and excitation of TPAI.

[0036] (5) Photodynamic properties of nanoparticles like Figure 5As shown, the ROS generation of each liposome group under 660 nm laser irradiation was detected using a DCFH fluorescent probe (Sigma-Aldrich, D6883). The fluorescence intensity changes of the Blank, CaCO3@Lipo, and CaCO3@Lipo-PyB groups were small, with almost no significant increase. In contrast, the fluorescence intensity of the CaCO3 / TPAI@Lipo and CaCO3 / TPAI@Lipo-PyB groups gradually increased with the extension of laser irradiation time, and after 1 min of irradiation, the relative fluorescence intensity (I / I0) of these two groups at 525 nm increased by 85-fold and 90-fold, respectively, indicating that both types of liposomes have significant photosensitizing ROS generation capabilities.

[0037] (6) Calcium release from nanoparticles like Figure 6 As shown, the calcium ion release behavior of nanoparticles under different pH conditions was determined using the o-cresolphthalein complex ketone colorimetric method. The results showed that the calcium release of the nanoparticles in Example 1 was 14.6% after 24 h under neutral conditions (pH 7.4), increasing to 26.8% under slightly acidic conditions (pH 6.5), and significantly accelerated under a more acidic environment (pH 5.0), with a cumulative release of 76.9% after 24 h. This result indicates that the nanoparticles exhibit typical acid-triggered calcium release characteristics.

[0038] 2. Antibacterial test (1) Nanoparticles inhibit bacterial acid production like Figure 7 As shown, when *Streptococcus mutans* was incubated in BHI medium containing 1% sucrose, the pH of the culture medium continuously decreased with prolonged incubation time, reaching approximately 4.5 after 12 hours, reflecting the bacteria's active acid-producing capacity. In contrast, after incubation with the nanoparticles of Example 1 for 2 hours followed by sterilization treatment with light for 20 minutes, the pH of the culture medium remained at approximately 7.0 throughout the entire incubation process because the number of viable bacteria was almost completely eliminated. This indicates that the nanoparticles of Example 1 of this invention can significantly inhibit bacterial acid production and block the formation of an acidic microenvironment after photoactivation.

[0039] (2) Nanoparticle biomembrane permeation experiment like Figure 8As shown, laser confocal microscopy was used to evaluate the permeation behavior of nanoparticles in *Streptococcus mutans* biofilms. The results showed that with prolonged incubation time, the red fluorescence signal inside the biofilm of both liposome treatment groups gradually increased, indicating that the permeation and accumulation of nanoparticles in the biofilm exhibited a significant time-dependent effect. Further comparison revealed that, compared to liposomes without charge-flipping properties, pH-responsive charge-flipping liposomes demonstrated stronger permeation and accumulation capabilities in the biofilm, proving their superior biofilm permeation performance.

[0040] (3) The killing and removal efficiency of nanoparticles against Streptococcus mutans biofilm like Figure 9 As shown, the bactericidal effect of each group of nanoparticles on *Streptococcus mutans* biofilm under light and non-light conditions was evaluated by the CFU counting method. The results showed that under non-light conditions, there was no significant difference in the number of CFUs in the biofilms of each group, indicating that the prepared nanoparticles had no significant dark toxicity to bacteria. After irradiation with a 660 nm laser, the CaCO3 / TPAI@Lipo group achieved approximately 98% viable bacterial kill, while the CaCO3 / TPAI@Lipo-PyB group, with its pH-responsive charge-reversal function, further improved its kill efficiency, reaching over 99.9%, demonstrating its significant advantage in photodynamic / photothermal synergistic antibacterial activity.

[0041] As shown in Figure 10, the removal efficiency of each group of nanoparticles on *Streptococcus mutans* biofilm was evaluated using crystal violet staining. The results showed that the CaCO3 / TPAI@Lipo group removed approximately 65% ​​of the biofilm, while the CaCO3 / TPAI@Lipo-PyB group, which possesses charge-flipping properties, further improved the biofilm removal rate to approximately 75%, indicating its superior ability to disrupt and remove mature biofilms.

[0042] The embodiments described herein are preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a dual pH-responsive charge-flipping-mineralization ion-releasing liposome formulation, characterized in that: Includes the following steps: S1. Preparation of mineralized functional nanomaterials S2. Preparation of dual pH-responsive charge-flipping-mineralized ion-releasing liposome nanoparticles a. Mix an ethanol solution containing mineralized nanomaterials with a chloroform solution of sodium 1,2-dioleoyl-sn-glycerol-3-phosphatidyl acid, sonicate in a water bath, centrifuge to collect the precipitate, and resuspend it in chloroform; wherein the mass ratio of mineralized nanomaterials to sodium 1,2-dioleoyl-sn-glycerol-3-phosphatidyl acid is (1-20):1; b. Add (R)-2,3-bis(palmitoyloxy)propyl(2-(trimethylammonium)ethyl)phosphate, DCPA, and DSPE-mPEG. 2000 Dissolved in chloroform, a lipid mixture was obtained; wherein (R)-2,3-bis(palmitoyloxy)propyl(2-(trimethylammonium)ethyl)phosphate, DCPA, and DSPE-mPEG were present. 2000 The mass ratio is 4 : (1.6-2.4) : (2.0-4.0); c. Mix the mixture obtained in step a with the mixture obtained in step b at room temperature and stir for 12-24 h. Add a dioxane solution containing TPAI and mix well. Remove chloroform by rotary evaporation to obtain a lipid membrane. Hydrate and centrifuge the lipid membrane to obtain a nanoparticle solution. Finally, dialyze to obtain a dual pH-responsive charge-flipping-mineralized ion-releasing liposome formulation. The mass ratio of mineralized functional nanomaterials, lipid mixture and TPAI is (0.24-0.32): 8.5: (0.18-0.23).

2. The method for preparing a dual pH-responsive charge-flipping-mineralization ion-releasing liposome formulation according to claim 1, characterized in that: In step S1, the mineralization functional nanomaterial is an inorganic nanomaterial capable of releasing calcium ions and / or promoting the formation of calcium phosphate mineralization; the mineralization functional nanomaterial is selected from calcium carbonate, calcium phosphate nanomaterials, bioactive glass, calcium peroxide and combinations thereof.

3. The method for preparing a dual pH-responsive charge-flipping-mineralization ion-releasing liposome formulation according to claim 2, characterized in that: The preparation method of calcium carbonate nanoparticles is as follows: Anhydrous calcium chloride is dissolved in water and diluted with anhydrous ethanol. The resulting mixture and ammonium bicarbonate are placed in two separate containers and reacted together at 35-45℃ for 6-12 hours. The reaction product is centrifuged to obtain calcium carbonate nanoparticles. The mass ratio of calcium chloride to ammonium bicarbonate is (1-3):

100.

4. The method for preparing a dual pH-responsive charge-flipping-mineralization ion-releasing liposome formulation according to claim 1, characterized in that: In step S2a, the ultrasonic water bath time is 20-30 min; the centrifugation conditions are: centrifugation at 8000-10000 rpm for 10-15 min.

5. The method for preparing a dual pH-responsive charge-flipping-mineralization ion-releasing liposome formulation according to claim 1, characterized in that: In step S2b, DSPE-mPEG 2000 Its molecular weight is 2000.

6. The method for preparing a dual pH-responsive charge-flipping-mineralization ion-releasing liposome formulation according to claim 1, characterized in that: In step S2c, the lipid membrane hydration treatment method is as follows: place the lipid membrane in water and ultrasonically bathe it at 45-50℃ for 1-3 minutes.

7. A dual pH-responsive charge-flipped-mineralized ion-releasing liposome formulation prepared by any of the preparation methods described in claims 1-6.

8. The use of the dual pH-responsive charge-flipping-mineralization ion-releasing liposome formulation of claim 7 in the preparation of a product for treating dental caries.

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