Fluoropolymer nanodroplets, methods of making and using the same

Abstract

The application belongs to the technical field of biological materials, and particularly relates to fluorine-containing polymer nanodroplets, a preparation method and application thereof. The nanodroplets are prepared by ultrasonic microemulsification of fluorine-containing polymer, perfluorocarbon and photosensitizer. The specific steps are as follows: perfluorocarbon, fluorine-containing polymer and photosensitizer are dissolved in an organic solvent, then ultrasonic treatment is performed, polyvinyl alcohol aqueous solution is added after uniform ultrasonic dispersion, ultrasonic treatment is performed again, the organic solvent is removed by rotary evaporation or ventilation and stirring evaporation after the mixture becomes emulsion, and fluorine-containing polymer nanodroplets are obtained by dialysis. The fluorine-containing polymer nanodroplets prepared by the application are loaded with perfluorocarbon by using the affinity between F-F for the first time. The fluorine-containing polymer nanodroplets can relieve bacterial biofilm and tumor tissue hypoxic microenvironment, and the photosensitizer in the nanodroplets can play a high-efficiency photodynamic therapy effect under the action of light, and can be used for bacterial killing and tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a fluoropolymer nanodroplet, its preparation method, and its application. Background Technology

[0002] Photodynamic therapy (PDT), a typical example of non-invasive treatment, has demonstrated excellent therapeutic effects in bacterial killing and tumor treatment. The three essential elements of PDT are photosensitizers, light, and oxygen. However, the inflammatory microenvironment, bacterial biofilm microenvironment, and tumor cell microenvironment are all hypoxic, and insufficient oxygen severely limits the efficiency of PDT. Therefore, alleviating the hypoxic state of these microenvironments is of great significance for PDT of bacteria and tumors. Perfluorinated carbon (PFCC) has extremely high oxygen solubility and is an important component in the manufacture of artificial blood. Furthermore, PFCC has good biocompatibility and is insoluble in biological fluids. Encapsulating PFCC with a polymer shell to form a protective layer can provide stability and facilitate its effectiveness. Although strategies for encapsulating contrast agents with nanoparticles already exist, the development of highly stable PFCC nanodroplets for efficient oxygen dissolution and the construction of a self-oxygenating PDT platform would greatly promote the treatment of diseases caused by hypoxic microenvironments. Summary of the Invention

[0003] To address the technical problem of poor photodynamic therapy caused by bacterial biofilms and hypoxia in the tumor microenvironment, this invention provides a method for preparing and applying fluoropolymer nanodroplets. To achieve the above objectives, this invention employs the following technical solutions:

[0004] A fluoropolymer nanodroplet, wherein the fluoropolymer nanodroplet is prepared by ultrasonic microemulsification of a fluoropolymer, perfluorinated carbon, and a photosensitizer;

[0005] The perfluorocarbon is at least one of perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluorobromooctane, perfluorononane, and perfluoronaphthane.

[0006] The photosensitizer is an aggregation-induced quenching (ACQ) photosensitizer or an aggregation-induced emission (AIE) photosensitizer;

[0007] The structural formula of the fluoropolymer is shown in Formula 1:

[0008]

[0009] Wherein, R1 is -H, -CH3, or -CH2CH3; R2 is -H, -CH3, or -CH2CH3; R3 is -H, -CH3, or -CH2CH3; Q is... One of the following, wherein R4 is -C6H5, -CH2C6H5, -CH2CH2COOH, -CH2CH2OH or -CH2OH; W is -C(CH3)3, -CHCN(CH3)2, -CH2C6H5 or -CCNCH3CH2CH2COOH; A is -O- or -NH-; B is -O- or -NH-; C is -O- or -NH-; D is -O- or -NH-; m = 2~200; n = 2~200; p = 2~200; a = 0~10; b = 0~4; c = 1~20; d = 0~10.

[0010] Furthermore, in Equation 1, a = 1, b = 2, c = 7, d = 7; Q is C6H5SS-; W is -CCNCH3CH2CH2COOH; A is -O-, B is -NH-, C is -NH-, and D is -O-. The structural formula corresponding to Equation 1 is shown in Equation 2.

[0011]

[0012] Wherein, R1 is -H, -CH3 or -CH2CH3; R2 is -H, -CH3 or -CH2CH3; R3 is -H, -CH3 or -CH2CH3; m = 2 to 200; n = 2 to 200; p = 2 to 200.

[0013] Furthermore, in Equation 2, R1 is -CH3, R2 is -CH3, and R3 is -CH3. The corresponding structural formula for Equation 2 is shown in Equation 3:

[0014]

[0015] Where m = 2 to 200; n = 2 to 200; p = 2 to 200.

[0016] Furthermore, the preparation method of the fluoropolymer includes the following steps:

[0017] In an organic solvent and in the presence of an initiator, fluorinated monomers, hydrophobic alkyl monomers, and macromolecular chain transfer agents are polymerized at 50–120 °C for 1–120 h in a vacuum environment. After the reaction is completed, the polymer is purified by post-treatment to obtain the fluorinated polymer.

[0018] Furthermore, the molar ratio of the initiator, macromolecular chain transfer agent, fluorinated monomer, and hydrophobic alkyl monomer is (0.1-0.5):1:(0.1-200):(0.1-200);

[0019] The organic solvent is at least one of 1,4-dioxane, dimethyl sulfoxide, dimethylformamide, tetrahydrofuran, and toluene; the preferred organic solvent is 1,4-dioxane:dimethyl sulfoxide = 3:1.

[0020] The initiator is at least one of azobisisobutyronitrile (AIBN), 4,4'-azobis(4-cyanopentanoic acid) (ACVA), azobisisoheptanenitrile (ABVN), and dimethyl azobisisobutyrate (AIBME);

[0021] The purification process specifically involves precipitating or ultrafiltration, dialysis, and lyophilizing the mixture after the reaction using low-polarity solutions such as petroleum ether, diethyl ether, cyclohexane, methyl tert-butyl ether, and ethyl acetate.

[0022] The preferred purification steps are as follows: Add a large amount of methyl tert-butyl ether to the obtained reaction solution for precipitation, discard the supernatant, dissolve the precipitate with dichloromethane, and precipitate again with petroleum ether. The volume of the undesirable solvent should be more than 5 times that of dichloromethane. Repeat this process three times and then dry to obtain a pink solid, which is Formula 1.

[0023] Furthermore, the structural formulas of the fluorinated monomer, the hydrophobic alkyl monomer, and the macromolecular chain transfer agent are shown in Formulas 4, 5, and 6:

[0024]

[0025] Wherein, R1 is -H, -CH3, or -CH2CH3; R2 is -H, -CH3, or -CH2CH3; R3 is -H, -CH3, or -CH2CH3; Q is... One of the following, wherein R4 is -C6H5, -CH2C6H5, -CH2CH2COOH, -CH2CH2OH or -CH2OH; W is -C(CH3)3, -CHCN(CH3)2, -CH2C6H5 or -CCNCH3CH2CH2COOH; A is -O- or -NH-; B is -O- or -NH-; C is -O- or -NH-; D is -O- or -NH-; m = 2 to 200; a = 0 to 10; b = 0 to 4; c = 1 to 20; d = 0 to 10.

[0026] Furthermore, the preparation method of the fluorinated monomer described in Formula 4 includes the following steps:

[0027] A fluorinated carboxylic acid sample, amino acrylate, triethylamine, and benzotriazole-N,N,N',N-tetramethylurea hexafluorophosphate (HBTU) were placed in a flask, dissolved in an organic solvent, sealed, and reacted for 1–120 h. After the reaction was completed, the resulting sample was purified by silica gel column chromatography to obtain a fluorinated monomer.

[0028] The reaction formula is:

[0029]

[0030] Wherein, R2 is -H, -CH3, or -CH2CH3; B is -O- or -NH-; C is -O- or -NH-; b = 0 to 4; c = 1 to 20;

[0031] The method for preparing the hydrophobic alkyl monomer described in Formula 5 includes the following steps:

[0032] Take an alkyl alcohol sample, enoyl chloride, and triethylamine in a flask, add anhydrous organic solvent, and react under sealed conditions for 1–48 h. After the reaction is complete, evaporate the solvent and separate the hydrophobic alkyl monomer using a silica gel column.

[0033] The reaction formula is:

[0034]

[0035] Wherein, R3 is -H, -CH3 or -CH2CH3, D is -O-, and d = 0 to 10;

[0036] The macromolecular chain transfer agent described in Formula 6 is prepared by a reversible addition-fragmentation chain transfer polymerization reaction, and the specific steps are as follows:

[0037] In an organic solvent, chain transfer agent QW, initiator, and N,N-dimethylenoic acid monomer are placed in a polymerization tube and reacted at 50–120 °C for 1–120 h under vacuum. After the reaction is completed, the macromolecular chain transfer agent is obtained by sedimentation purification.

[0038] The reaction formula is:

[0039]

[0040] Where R1 is -H, -CH3, or -CH2CH3; Q is One of them, wherein R4 is -C6H5, -CH2C6H5, -CH2CH2COOH, -CH2CH2OH or -CH2OH; W is -C(CH3)3, -CHCN(CH3)2, -CH2C6H5 or -CCNCH3CH2CH2COOH; A is -O- or -NH-; a = 0 to 10.

[0041] Furthermore, the molar ratio of the fluorocarboxylic acid sample, aminoacrylate, triethylamine and benzotriazole-N,N,N',N-tetramethylurea hexafluorophosphate is 1:(1-5):(1-10):(1-10);

[0042] The organic solvent is DMF, and the amount used is 0.1-20 mL;

[0043] When R2 is -CH3, B is -NH-, C is -NH-, b=2, and c=7, the fluorocarboxylic acid sample is heptadecanodecanoic acid, and the amino acrylate is N-(3-aminopropyl)methacrylamide hydrochloride.

[0044] The purification process specifically involves: extracting the obtained sample with ethyl acetate and sodium chloride solution or water, and then separating and purifying it using a silica gel column to obtain the fluorine-containing monomer shown in Formula 4; the eluent used for column separation is ethyl acetate: petroleum ether = 1~100:1 (V:V), preferably 10:1.

[0045] Furthermore, the molar ratio of the alkyl alcohol sample, enoyl chloride, and triethylamine is 1:(1-5):(1-5); preferably, the molar ratio of the alkyl alcohol sample, enoyl chloride, and triethylamine is 1:1.2:2.

[0046] The anhydrous organic solvent is dichloromethane or tetrahydrofuran, and the amount used is 10-200 mL.

[0047] When R3 = -CH3 and d = 7, the alkyl alcohol sample is nonanol, and the methacryloyl chloride is methacryloyl chloride;

[0048] The purification process specifically involves: extracting the obtained reaction solution with dichloromethane and sodium bicarbonate solution or water, and then separating and purifying it using a silica gel column to obtain the hydrophobic alkyl monomer shown in Formula 5; the eluent used for column separation is petroleum ether:dichloromethane = 1~100:1 (V:V), preferably 10:1.

[0049] Furthermore, the molar ratio of the chain transfer agent QW, N,N-dimethacrylic acid monomer, and initiator is 1:(1-200):(0.1-0.5); preferably, the molar ratio of the chain transfer agent QW, N,N-dimethacrylic acid monomer, and initiator is 1:30:0.2.

[0050] The organic solvent is at least one of 1,4-dioxane, dimethyl sulfoxide, dimethylformamide, tetrahydrofuran, and toluene;

[0051] When Q is C6H5SS-, W is -CCNCH3CH2CH2COOH, R1 is -CH3, A is -O-, and a = 1, the chain transfer agent is 4-cyano-4-(thiobenzoyl)valerate, and the N,N-dimethylenoic acid monomer is ethyl 2-dimethylaminomethacrylate.

[0052] The purification process specifically involves precipitating or ultrafiltration, dialysis, and lyophilizing the mixture after the reaction using one or more solutions selected from petroleum ether, diethyl ether, cyclohexane, methyl tert-butyl ether, and ethyl acetate.

[0053] The preferred purification procedure is to pour the obtained reaction solution into petroleum ether for precipitation, then centrifuge, dissolve the resulting precipitate in dichloromethane, and then precipitate it again with petroleum ether. The volume of the unsuitable solvent should be more than 5 times that of the dichloromethane. This process is repeated three times. The resulting solid is then dried under vacuum to obtain the purified macromolecular chain transfer agent.

[0054] This invention also provides a method for preparing fluoropolymer nanodroplets, the method comprising the following steps:

[0055] Perfluorinated carbon, fluoropolymers, and photosensitizers are dissolved in an organic solvent in a certain proportion, and then sonicated. After the mixture is sonicated and evenly dispersed, an aqueous solution of polyvinyl alcohol (PVA) is added, and the mixture is sonicated again until it becomes an emulsion. The organic solvent is then removed by rotary evaporation or aeration stirring. Finally, dialysis is performed to remove unloaded small molecules to obtain fluoropolymer nanodroplets.

[0056] The organic solvent is at least one of dichloromethane, tetrahydrofuran, and trichloromethane;

[0057] The ultrasonic power is 50-1500W;

[0058] The mass concentration of the polyvinyl alcohol aqueous solution is 0.5-5%;

[0059] The volume ratio of the organic solvent to the polyvinyl alcohol aqueous solution is 1:5-20; the ratio of the amount of perfluorinated carbon, fluoropolymer, and photosensitizer is 0.01-1 mL:0.5-10 mg:0.1-5 mg.

[0060] The dialysis used a molecular weight cutoff of 1–10 kDa.

[0061] The present invention also provides an application of fluoropolymer nanodroplets for efficient oxygen loading and for the preparation of antibacterial and tumor therapeutic products.

[0062] The fluoropolymer nanodroplets have a high perfluorinated carbon content and can maintain a high oxygen content for a long time after oxygen is introduced. Furthermore, they exhibit a strong photodynamic therapy effect after light irradiation, effectively killing bacteria.

[0063] Principle: A unique FF hydrophobic interaction exists between the fluorine atoms in the fluoropolymer and the fluorine atoms in perfluorinated carbon, enabling the efficient and stable formation of nanodroplets. The polymer is on the outer layer, and the perfluorinated carbon is on the inner layer. Due to the extremely high solubility of perfluorinated carbon in oxygen, the nanodroplets, when oxygen is introduced, can serve as a self-oxygenating therapeutic platform, maintaining a high oxygen content for an extended period. The hydrophilic portion of the nanodroplets can be protonated under acidic conditions, increasing their positive charge and facilitating adhesion to negatively charged bacteria. After light treatment, the photosensitizer in the core exerts a photodynamic effect, resulting in highly efficient bactericidal action.

[0064] Compared with the prior art, the present invention has the following advantages:

[0065] (1) The fluoropolymer nanodroplets prepared by this invention are the first to propose using the affinity between F and F to load perfluorinated carbon.

[0066] (2) Fluoropolymer nanodroplets can efficiently load oxygen, which is beneficial to alleviate the hypoxic conditions of inflammation, bacterial biofilms, and tumor microenvironment.

[0067] (3) Fluoropolymer nanodroplets can serve as a self-oxygenated therapy platform, exerting a highly efficient photodynamic therapy effect under light conditions, and can be used for bacterial killing and tumor treatment. Attached Figure Description

[0068] Figure 1 This is the 1H NMR spectrum of a fluorine monomer;

[0069] Figure 2 This is the 1H NMR spectrum of the n-nonyl alcohol monomer;

[0070] Figure 3 This is the 1H NMR spectrum of a macromolecular chain transfer agent;

[0071] Figure 4 This is the 1H NMR spectrum of a fluoropolymer;

[0072] Figure 5 These are particle size diagrams and transmission electron microscope (TEM) and scanning electron microscope (SEM) images of fluoropolymer nanodroplets.

[0073] Figure 6 These are the absorption spectra of fluoropolymer nanodroplets and the absorption standard curves of photosensitizers;

[0074] Figure 7 This is a gas chromatography result of the perfluorinated carbon content in fluoropolymer nanodroplets;

[0075] Figure 8 This is a graph showing the oxygen content detection of fluoropolymer nanodroplets;

[0076] Figure 9 This is a test diagram of the photodynamic properties of fluoropolymer nanodroplets;

[0077] Figure 10 This is a diagram showing the antibacterial properties of fluoropolymer nanodroplets against Streptococcus mutans (S. mutans);

[0078] Figure 11 This is a graph showing the anti-biofilm performance of fluoropolymer nanodroplets against biofilms formed by Streptococcus mutans (S. mutans);

[0079] Figure 12 This is a scanning electron microscope image showing the disruption of biofilms formed by fluoropolymer nanodroplets by Streptococcus mutans (S. mutans). Detailed Implementation

[0080] The raw materials used in the examples are described below:

[0081] N-(3-aminopropyl)methacrylamide hydrochloride, heptadecanofluoroundecanoic acid, n-nonanol, perfluorooctane, and 9,10-anthratridiyl-bis(methylene)dicarboxylic acid were purchased from Macklin Biotechnology; benzotriazole-N,N,N',N-tetramethylurea hexafluorophosphate (HBTU), azobisisobutyronitrile, and ethyl 2-dimethylaminomethacrylate were purchased from Aladdin Biotechnology; methacryloyl chloride was purchased from Energy. Chemical Company; chain transfer agent 4-cyano-4-(thiobenzoylthio)valerate was purchased from Bid Pharmaceutical; mesitylene trimethoxybenzene was purchased from Alpha Company; triethylamine was purchased from Shandong Xiya Chemical Industry Co., Ltd.; dimethyl sulfoxide and N,N-dimethylformamide were purchased from Tianjin Dongfangzheng Company; dichloromethane, petroleum ether, ethyl acetate and other reagents were all analytical grade and purchased from Tianjin Beichen Fangzheng Reagent Company; BHI broth culture medium was purchased from Guangdong Huankai Biotechnology Co., Ltd.; ultrapure water used in the experiment was prepared by the Milli-Q intelligent ultrapure water system.

[0082] Example 1

[0083] Preparation of fluorinated monomers

[0084] N-(3-aminopropyl)methacrylamide hydrochloride (APMA, 164 mg, 0.896 mmol) and triethylamine (0.16 mL, 2.33 mmol) were weighed and dissolved in 2 mL of N,N-dimethylformamide. After stirring for 30 min, heptadecanofluoroundecanoic acid (440 mg, 0.894 mmol), benzotriazole-N,N,N',N-tetramethylurea hexafluorophosphate (HBTU, 1200 mg, 3.164 mmol), and 2 mL of N,N-dimethylformamide were added and stirred for 3 days. After the reaction was completed, the mixture was extracted with ethyl acetate and ultrapure water. The upper organic phase was evaporated by rotary evaporation and purified by silica gel column chromatography. The fluorinated monomer (FMA) was eluted with ethyl acetate:petroleum ether (V:V) = 9:1 to obtain a white solid (0.37 g, yield: 61.26%).

[0085] The reaction formula is:

[0086]

[0087] The fluorine-containing monomer FMA prepared in Example 1 was analyzed by proton nuclear magnetic resonance spectroscopy (e.g., Figure 1 As can be seen from the figure, the proportion of hydrogen in the molecular formula is the same as the peak area ratio in the corresponding hydrogen NMR spectrum, indicating that FMA was successfully synthesized.

[0088] Example 2

[0089] Preparation of hydrophobic alkyl monomers

[0090] Weigh 1.2 mL of nonanol (6.93 mmol), 1.95 mL of triethylamine (13.835 mmol), and 20 mL of anhydrous dichloromethane into a round-bottom flask. Add 0.81 mL of methacryloyl chloride (8.322 mmol) and 20 mL of anhydrous dichloromethane into a constant-pressure dropping funnel. After assembling the mixture, add the methacryloyl chloride dropwise to the round-bottom flask under nitrogen protection and stir in an ice-water bath for 24 h. After the reaction is complete, extract with dichloromethane and ultrapure water. The lower organic phase is collected by rotary evaporation and purified by silica gel column chromatography. Elute with petroleum ether:dichloromethane (V:V) = 9:1 to obtain a hydrophobic alkyl monomer (RMA) as an oily liquid (0.75 g, yield: 40.11%).

[0091] The reaction formula is:

[0092]

[0093] The hydrophobic alkyl monomer (RMA) prepared in Example 2 was analyzed by proton nuclear magnetic resonance spectroscopy (e.g., Figure 2 As can be seen from the figure, the proportion of hydrogen in the molecular formula is the same as the peak area ratio in the corresponding hydrogen NMR spectrum, indicating that RMA was successfully synthesized.

[0094] Example 3

[0095] Preparation of poly(2-dimethylaminomethyl methacrylate) PDMAEMA macromolecular chain transfer agent

[0096] Chain transfer agent 4-cyano-4-(thiobenzoylthio)pentanoic acid (CTA, 104 mg, 0.372 mmol), ethyl 2-dimethylaminomethacrylate (DMAEMA, 1730 mg, 11 mmol), and azobisisobutyronitrile (AIBN, 12.28 mg, 0.074 mmol) were dissolved in 1 mL of 1,4-dioxane in a polymerization tube. The polymerization tube was placed in liquid nitrogen for freezing, then evacuated with an oil pump for 3 min. The polymerization tube was then sealed and allowed to return to room temperature to thaw the reaction mixture. This freeze-thaw cycle was repeated three times to achieve a vacuum state, and finally, the tube was sealed. The polymerization reaction was terminated by stirring in an oil bath at 70 °C for 24 h with liquid nitrogen. The polymerization tube was then opened, and the reaction mixture was allowed to settle in petroleum ether. After centrifugation at 3500 rpm for 5 min, the supernatant was discarded, and the precipitate was dissolved in dichloromethane and settled with a large amount of petroleum ether. This process was repeated three times, with the volume of petroleum ether being 10 times that of dichloromethane. The final product was dried overnight at room temperature in a vacuum drying oven to obtain a red viscous solid, namely the macromolecular chain transfer agent (1005 mg, yield 55%).

[0097] The reaction formula is:

[0098]

[0099] The macromolecular chain transfer agent PDMAEMA prepared in this embodiment was subjected to 1H NMR spectroscopy (e.g., Figure 3 (a) Analysis shows that, by comparing the integral area of ​​the protons on the benzene ring in the chain transfer agent at positions above 7.0 ppm with the integral area of ​​the protons on the N-methyl group in ethyl 2-dimethylaminomethacrylate at 2.3 ppm, the degree of polymerization of the macromolecular chain transfer agent PDMAEMA prepared in this example is found to be 44, and its molecular formula is PDMAEMA. 44 .

[0100] Following the same method, chain transfer agent 4-cyano-4-(thiobenzoylthio)pentanoic acid (CTA, 52 mg, 0.186 mmol), ethyl 2-dimethylaminomethacrylate (DMAEMA, 857 mg, 5.451 mmol), and azobisisobutyronitrile (AIBN, 6 mg, 0.036 mmol) were dissolved in 1 mL of 1,4-dioxane in a polymerization tube. The mixture was then processed according to the above steps, and after purification, a red solid (524 mg, yield 57.65%) was obtained. The results were analyzed using 1H NMR (e.g., 1H NMR spectroscopy). Figure 3(b) Analysis revealed that the polymer of the macromolecular chain transfer agent PDMAEMA prepared in this embodiment was 36, and its molecular formula was PDMAEMA. 36 .

[0101] Example 4

[0102] Preparation of fluoropolymers

[0103] The macromolecular chain transfer agent PDMAEMA prepared in Example 3 44 The reaction mixture (300 mg, 0.041 mmol) was dissolved in 2 mL of 1,4-dioxane and 0.75 mL of dimethyl sulfoxide in a polymerization tube along with the fluorinated monomer FMA (660 mg, 1.07 mmol) prepared in Example 1, the hydrophobic alkyl monomer RMA (107.2 mg, 0.505 mmol) prepared in Example 2, and azobisisobutyronitrile (AIBN) (1.67 mg, 0.010 mmol). The polymerization tube was frozen in liquid nitrogen, then evacuated using an oil pump. The tube was sealed and allowed to return to room temperature to thaw the reaction mixture. This freeze-thaw cycle was repeated three times to achieve a vacuum state, and finally, the tube was sealed. After stirring in an oil bath at 70°C for 24 h, the polymerization reaction was terminated with liquid nitrogen. The polymerization tube was opened, and the resulting mixture was precipitated in petroleum ether. After centrifugation, the supernatant was discarded, and the precipitate was dissolved in dichloromethane and precipitated with a large amount of petroleum ether. This process was repeated three times, with the volume of petroleum ether being 10 times that of dichloromethane. The final product was dried overnight at room temperature in a vacuum drying oven to obtain a red solid (450 mg, yield 58.66%), which is the fluoropolymer PDMAEMA-bP (FMA-co-RMA).

[0104] The reaction formula is:

[0105]

[0106] The fluoropolymer PDMAEMA-bP (FMA-co-RMA) prepared in Example 4 was subjected to 1H NMR spectroscopy (e.g., Figure 4 (a) Analysis shows that, by comparing the integral area of ​​hydrogen at position a in DMAEMA at 2.3 ppm, the integral area of ​​hydrogen at positions c and d in FMA at 3.2 ppm, and the integral area of ​​hydrogen at position d in RMA at 4.1 ppm, the degree of polymerization of FMA prepared in Example 1 is 9, the degree of polymerization of RMA is 16, and the molecular formula of the fluoropolymer is PDMAEMA. 44 -bP(FMA9-co-RMA 16 ).

[0107] Following the same method, the macromolecular chain transfer agent PDMAEMA prepared in Example 3 was... 44(300 mg, 0.041 mmol) and the fluorinated monomer FMA (625 mg, 1.014 mmol) prepared in Example 1, the hydrophobic alkyl monomer RMA (107.2 mg, 0.505 mmol) prepared in Example 2, and azobisisobutyronitrile (AIBN) (1.67 mg, 0.010 mmol) were dissolved in 1.5 mL of 1,4-dioxane and 0.3 mL of dimethyl sulfoxide in a polymerization tube. The mixture was processed according to the above steps, purified, and yielded a red solid (425 mg, 46% yield). The NMR spectrum (e.g., 1H NMR) was then analyzed. Figure 4 (b) Analysis showed that the degree of polymerization of FMA prepared in this embodiment was 11, and the degree of polymerization of RMA was 10. Its molecular formula is PDMAEMA. 44 -bP(FMA 11 -co-RMA 10 ).

[0108] Following the same method, the macromolecular chain transfer agent PDMAEMA prepared in Example 3 was... 36 (130 mg, 0.022 mmol) fluorinated monomer FMA (271 mg, 0.44 mmol) prepared in Example 1, hydrophobic alkyl monomer RMA (58.7 mg, 0.277 mmol) prepared in Example 2, and azobisisobutyronitrile (AIBN) (0.73 mg, 0.004 mmol) were dissolved in 1.5 mL of 1,4-dioxane and 0.5 mL of dimethyl sulfoxide in a polymerization tube. The mixture was processed according to the above steps, purified, and yielded a red solid (230 mg, 50% yield). The NMR spectrum (e.g., 1H NMR) was analyzed. Figure 4 (c) Analysis showed that the degree of polymerization of FMA prepared in this embodiment was 4, and the degree of polymerization of RMA was 6. Its molecular formula is: PDMAEMA. 36 -bP(FMA4-co-RMA6).

[0109] Example 5

[0110] Preparation of fluoropolymer nanodroplets

[0111] 3mg PDMAEMA 44 -bP(FMA9-co-RMA 161 mg of photosensitizer (TPA-BZ) (according to the reference, aggregation-induced quenching (ACQ) photosensitizer (JAm Chem Soc, 2020, 142(39), 16632-16643)) was dissolved in 1 mL of dichloromethane, and then 100 μL of perfluorobromooctane (PFOB) was added. The mixture was ultrasonically treated using a liquid crystal ultrasonic processor (900 W, 3 s ON 4 s OFF, 11 min). After the mixture was ultrasonically dispersed evenly, 8 mL of 1% polyvinyl alcohol aqueous solution (PVA) (10 g / L) was added. The mixture was then ultrasonically treated again using an ultrasonic processor (900 W, 3 s ON 4 s OFF, 11 min) to obtain an orange milky liquid. Finally, after removing the dichloromethane by rotary evaporation, the mixture was dialyzed with ultrapure water using a 3.5 kDa dialysis bag for 8 h. The resulting nanoparticle solution was abbreviated as PFC / TPA@FNPs.

[0112] Example 6

[0113] Analysis of fluoropolymer nanodroplets using dynamic light scattering particle size analyzer (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM).

[0114] The prepared PFC / TPA@FNPs were diluted to 100 μg / mL, and the particle size distribution of the material was analyzed using a four-way quartz cuvette. Figure 5 As shown in (a), the hydrated particle size of PFC / TPA@FNPs is in the range of 100–200 nm. A copper mesh was gently held with electron microscope tweezers and rinsed several times in the PFC / TPA@FNPs solution to ensure as much sample as possible adhered to the mesh. The sample-coated copper mesh was then placed in an EP tube and immersed in liquid nitrogen under vacuum using an oil pump. The morphology and size of the material were then observed using a JEM-2100F transmission electron microscope (TEM) and a Zeiss Gemini 300 scanning electron microscope (SEM). Figure 5 (b) and (c) are transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of the synthesized PFC / TPA@FNPs, respectively. The results show that the morphology of PFC / TPA@FNPs is a spherical shape with cavities of about 100 nm, which is consistent with the results of DLS detection data.

[0115] Example 7

[0116] UV-Vis absorption spectrum of fluoropolymer nanodroplets

[0117] Fluorescence spectroscopy analysis of fluoropolymer nanodroplets PFC / TPA@FNPs, TPA-BZ and PDMAEMA 44 -bP(FMA 9- co-RMA 16 The absorption spectrum of ) is as follows Figure 6 As shown in (a), the synthesized PFC / TPA@FNPs contain the characteristic absorption peak of TPA-BZ at 445 nm. The absorption standard curve of TPA-BZ was calculated based on the following steps: TPA-BZ was dissolved in dimethyl sulfoxide to prepare liquids with concentrations of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, and 3.125 μg / mL. The absorption curves were measured using a UV-Vis spectrophotometer. The standard curve for TPA-BZ was then plotted based on the absorbance at 445 nm. The standard curve is y = 0.03782 + 11.0172x (e.g., ...). Figure 6 (b) shows that y is the absorbance value and x is the corresponding concentration of TPA-BZ. The amount of TPA-BZ encapsulated in 100 μg / mL PFC / TPA@FNPs is 3.12 μg, and the photosensitizer loading rate of fluoropolymer nanodroplets PFC / TPA@FNPs can reach 94.4%.

[0118] Gas chromatograph analysis of perfluorinated carbon content

[0119] First, dissolve 0.25 μL, 0.5 μL, 2.5 μL, 5 μL, 10 μL, 15 μL, 20 μL, and 30 μL of perfluorobromooctane and 10 mg of mesitylene in 200 μL of 1,1.1,3,3-pentafluorobutane, respectively. Inject 1 μL of each solution into a gas chromatograph. Plot the peak areas to form a standard curve. The standard curve is Y = 0.54929x - 0.15759 (e.g., ...). Figure 7 (as shown in (a)), where y is the ratio of the peak areas of 15.37 × perfluorobromooctane and mesitylene trimethoxybenzene, and x is the corresponding volume of perfluorobromooctane. 50 μL of 0.8 mg / mL LPFC / TPA@FNPs (with 3 mg PDMAEMA) 44 -bP(FMA9-co-RMA 16 1 mg of photosensitizer (TPA-BZ) and 300 μL of perfluorobromooctane (PFOB, prepared using the method in Example 5) were dissolved in 100 μL of acetonitrile and vortexed for 120 s. Then, 200 μL of 1.1.1.3.3-pentafluorobutane was added, and the mixture was vortexed for 300 s. The mixture was centrifuged (4 °C, 1500 g, 2 min) and frozen overnight at -20 °C. Finally, 150 μL of the lower phase solution was taken and 10 mg of trimethoxybenzene was dissolved in it. 1 μL of the solution was injected into the gas chromatograph, and the peak area was used to calculate the standard curve. It was found that the volume of PFOB contained in 0.8 mg / mL PFC / TPA@FNPs was 96.58 μL, and the encapsulation efficiency was 32.195% (e.g., ...). Figure 7 (b) is shown.

[0120] Oxygen content detection and analysis of fluoropolymer nanodroplets

[0121] The oxygen-carrying capacity of nanodroplets was measured using a portable dissolved oxygen meter. 3 mL of 200 μg / mL PFC / TPA@FNPs and ultrapure water were each purged with oxygen for 15 min. Then, 0.5 mL of each mixture was added to 2.5 mL of anoxic water. The oxygen content of the mixture was measured using the portable dissolved oxygen meter over 20 min at 37°C. In the control group, 3 mL of 200 μg / mL PFC / TPA@FNPs and ultrapure water were each purged with nitrogen for 15 min. Then, 0.5 mL of each mixture was added to 2.5 mL of anoxic water. The oxygen content of the mixture was measured using the portable dissolved oxygen meter over 20 min at 37°C. Figure 8 As shown, this indicates that PFC / TPA@FNPs have better oxygen-carrying capacity.

[0122] Photodynamic performance testing of fluoropolymer nanodroplets

[0123] 9,10-Anthracenediyl-bis(methylene)dicarboxylic acid (ABDA) singlet oxygen fluorescent probe is an indicator for determining singlet oxygen. This reaction is detected by spectrophotometric recording of the decrease in optical density at 400 nm. 1.99 mL of 25 μg / mPFC / TPA@FNPs was added to 0.01 mL of dimethyl sulfoxide as a baseline. The UV absorbance of the mixture of 1.99 mL of 25 μg / mPFC / TPA@FNPs and 0.01 mL of 10 mLPFC / TPA@FNPs was measured by irradiation with a 445 nm blue light lamp for different durations. The above operation was repeated with photosensitizer (TPA-BZ) and oxygen-treated PFC / TPA@FNPs to obtain the photodynamic performance test graph (e.g., PFC / TPA@FNPs). Figure 9 As shown in the figure, this indicates that oxygen-carrying PFC / TPA@FNPs have stronger photodynamic properties.

[0124] Example 8

[0125] Antibacterial properties of fluoropolymer nanodroplets

[0126] The minimum bactericidal concentration (MBC) of the material was determined using the microbroth dilution method to assess its antibacterial properties. Gram-positive Streptococcus mutans (ATCC 25175) was selected as the model bacterium. 200 μL of 25 μg / mL material was added to each well of a 96-well plate, and 100 μL of PBS was added to the remaining wells sequentially. The plates were then serially diluted to obtain different concentration gradients (25 μg / mL - 1.5625 μg / mL). The absorbance (OD600) of the bacterial suspension at 600 nm was measured using UV-Vis absorption spectroscopy. The suspensions were then diluted with PBS to obtain the desired bacterial concentration (1 × 10⁻⁶).6 (CFU / mL). Add 100 μL of bacterial suspension to the diluted material, shake well, and incubate in a 37°C shaker for 30 min. Irradiate with a 445 nm blue light lamp and then dilute 10. 2 Then take 100 μL and coat it onto the plate. Figure 10 (a) is a plate diagram of the antibacterial experiment of PFC / TPA@FNPs against S. mutans. Figure 10 (b)(c) shows the count statistics of the antibacterial test plates of PFC / TPA@FNPs against S. mutans. As can be seen from the figure, the MBC value of the fluoropolymer nanodroplets against S. mutans is 3.125 μg / mL.

[0127] Example 9

[0128] Anti-biofilm properties of fluoropolymer nanodroplets

[0129] The anti-biofilm properties of the material were assessed using the micro-broth dilution method. Gram-positive Streptococcus mutans (ATCC 25175) was selected as the model bacterium. The absorbance (OD600) of the bacterial suspension at 600 nm was measured using UV-Vis absorption spectroscopy. The suspension was then diluted with PBS to obtain the desired bacterial concentration (1×10⁻⁶). 6 CFU / mL), 200 μL of diluted bacterial suspension was added to each well of a 96-well plate and incubated at 37°C for 48 h to form a biofilm. After washing the airborne bacteria with PBS, 200 μL of oxygen-fluffed PFC / TPA@FNPs (200 μg / mL) was added, shaken well, and incubated in a 37°C shaker for 30 min. After irradiation with a 445 nm blue light lamp, the airborne bacteria were washed with PBS and dried at room temperature. Then, 200 μL of PBS was added again, and the mixture was sonicated and diluted 10⁻⁶ times. 4 100 μL of the sample was plated, and PBS and no-light groups were added as control groups. Figure 11 The figure shows the antibacterial test results of PFC / TPA@FNPs against S. mutans. As can be seen from the figure, the concentration of fluoropolymer nanodroplets at 200 μg / mL has anti-biofilm properties, and the anti-biofilm properties are enhanced after oxygenation.

[0130] After gradient dehydration of healthy *S. mutans* biofilms and those treated with the material, bacterial morphology was observed using scanning electron microscopy. The results showed that the morphology of *S. mutans* changed after material treatment (e.g., ...). Figure 12 (As shown).

[0131] In summary, the fluoropolymer nanodroplets obtained by this invention can alleviate the hypoxic state of bacterial biofilms and tumor tissue microenvironments. At the same time, the photosensitizer in the nanodroplets can exert a highly efficient photodynamic therapy effect under light irradiation, thereby achieving the effects of bacterial killing and tumor treatment, and has good application prospects in the biomedical field.

[0132] Contents not described in detail in this specification are prior art known to those skilled in the art. Although illustrative specific embodiments of the invention have been described above to facilitate understanding by those skilled in the art, it should be understood that the invention is not limited to the scope of the specific embodiments. Various modifications are readily apparent to those skilled in the art as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims, and all inventions utilizing the concept of this invention are protected.

Claims

1. A fluoropolymer nanodroplet, characterized in that: The fluoropolymer nanodroplets were prepared by ultrasonic microemulsification of fluoropolymer, perfluorinated carbon, and photosensitizer. Furthermore, there is a unique FF hydrophobic interaction between the fluorine atoms in the fluoropolymer and the fluorine atoms in the perfluorinated carbon, which can efficiently and stably form nanodroplets; The perfluorocarbon is at least one selected from perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluorobromooctane, perfluorononane, and perfluoronaphthane. The photosensitizer is an aggregation-induced emission type photosensitizer; The structural formula of the fluoropolymer is shown in Formula 1: ； Where R1 is -H or -CH3; R2 is -H or -CH3; R3 is -H or -CH3; Q is One of the following, wherein R4 is -C6H5, -CH2C6H5, -CH2CH2COOH, -CH2CH2OH, or -CH2OH; W is -C(CH3)3, -CHCN(CH3)2, -CH2C6H5, or -CCNCH3CH2CH2COOH; A is -O-; B is -NH-; C is -NH-; D is -O-; m=2~200; n=2~200; p=2~200; a=1; b=2; c=7; d=7; The preparation method of the fluoropolymer includes the following steps: In an organic solvent and in the presence of an initiator, fluorinated monomers, hydrophobic alkyl monomers, and macromolecular chain transfer agents are polymerized at 50–120 °C for 1–120 h in a vacuum environment. After the reaction is completed, the polymer is purified by post-treatment to obtain the fluorinated polymer.

2. The fluoropolymer nanodroplet according to claim 1, characterized in that: In Equation 1, Q is C6H5SS-; W is -CCNCH3CH2CH2COOH; the corresponding structural formula of Equation 1 is shown in Equation 2: ； Wherein, R1 is -H or -CH3; R2 is -H or -CH3; R3 is -H or -CH3; m = 2 to 200; n = 2 to 200; p = 2 to 200.

3. The fluoropolymer nanodroplet according to claim 2, characterized in that: In Equation 2, R1 is -CH3, R2 is -CH3, and R3 is -CH3. The corresponding structural formula for Equation 2 is shown in Equation 3. ； Where m = 2~200; n = 2~200; p = 2~200.

4. The fluoropolymer nanodroplet according to claim 1, characterized in that: The molar ratio of the initiator, macromolecular chain transfer agent, fluorinated monomer and hydrophobic alkyl monomer is (0.1-0.5):1:(0.1-200):(0.1-200); The organic solvent is at least one of 1,4-dioxane, dimethyl sulfoxide, dimethylformamide, tetrahydrofuran, and toluene; The initiator is at least one of azobisisobutyronitrile, 4,4'-azobis(4-cyanopentaic acid), azobisisoheptanenitrile, and dimethyl azobisisobutyrate; The purification process specifically involves precipitating or ultrafiltration, dialysis, and lyophilizing the mixture after the reaction using one or more solutions selected from petroleum ether, diethyl ether, cyclohexane, methyl tert-butyl ether, and ethyl acetate.

5. The fluoropolymer nanodroplet according to claim 1, characterized in that: The structural formulas of the fluorinated monomer, the hydrophobic alkyl monomer, and the macromolecular chain transfer agent are shown in Formulas 4, 5, and 6. ； Where R1 is -H or -CH3; R2 is -H or -CH3; R3 is -H or -CH3; Q is One of the following, wherein R4 is -C6H5, -CH2C6H5, -CH2CH2COOH, -CH2CH2OH or -CH2OH; W is -C(CH3)3, -CHCN(CH3)2, -CH2C6H5 or -CCNCH3CH2CH2COOH; A is -O-; B is -NH-; C is -NH-; D is -O-; m=2~200; a=1; b=2; c=7; d=7.

6. The fluoropolymer nanodroplet according to claim 5, characterized in that: The preparation method of the fluorinated monomer described in Formula 4 includes the following steps: Take the fluorocarboxylic acid sample, aminoacrylate, triethylamine, and benzotriazole-N,N,N',N-tetramethylurea hexafluorophosphate in a flask, add an organic solvent to dissolve them, seal the flask and react for 1 to 120 hours. After the reaction is completed, purify the obtained sample by silica gel column chromatography to obtain the fluorinated monomer. The reaction formula is: ； Where R2 is -H or -CH3, B is -NH-, C is -NH-, b=2, c=7; The preparation method of the hydrophobic alkyl monomer described in Formula 5 includes the following steps: Take an alkyl alcohol sample, enoyl chloride, and triethylamine in a flask, add anhydrous organic solvent, and react under sealed conditions for 1–48 h. After the reaction is complete, evaporate the solvent and separate the hydrophobic alkyl monomer using a silica gel column. The reaction formula is: ； Where R3 is -H or -CH3, D is -O-, and d=7; The macromolecular chain transfer agent described in Formula 6 is prepared by a reversible addition-fragmentation chain transfer polymerization reaction, specifically through the following steps: In an organic solvent, chain transfer agent QW, initiator, and N,N-dimethylenoic acid monomer are placed in a polymerization tube and reacted at 50–120 °C for 1–120 h under vacuum. After the reaction is completed, the macromolecular chain transfer agent is obtained by sedimentation purification. The reaction formula is: ； Where R1 is -H or -CH3; Q is One of them, wherein R4 is -C6H5, -CH2C6H5, -CH2CH2COOH, -CH2CH2OH or -CH2OH; W is -C(CH3)3, -CHCN(CH3)2, -CH2C6H5 or -CCNCH3CH2CH2COOH; A is -O-; a=1.

7. The fluoropolymer nanodroplet according to claim 6, characterized in that: The molar ratio of the fluorocarboxylic acid sample, aminoacrylate, triethylamine and benzotriazole-N,N,N',N-tetramethylurea hexafluorophosphate is 1:(1-5):(1-10):(1-10); The organic solvent is DMF, and the amount used is 0.1-20 mL; When R2 is -CH3, B is -NH-, C is -NH-, b=2, and c=7, the fluorocarboxylic acid sample is heptadecanodecanoic acid, and the amino acrylate is N-(3-aminopropyl)methacrylamide hydrochloride. The purification process specifically involves: extracting the obtained sample with ethyl acetate and sodium chloride solution or water, and then separating and purifying it using a silica gel column to obtain the fluorine-containing monomer shown in Formula 4; the eluent used for column separation is ethyl acetate:petroleum ether with a volume ratio of 1 to 100:

1.

8. The fluoropolymer nanodroplet according to claim 6, characterized in that: The molar ratio of the alkyl alcohol sample, enoyl chloride, and triethylamine is 1:(1-5):(1-5); The anhydrous organic solvent is dichloromethane or tetrahydrofuran, and the amount used is 10-200 mL. When R3=-CH3 and d=7, the alkyl alcohol sample is nonanol, and the methacryloyl chloride is methacryloyl chloride; The purification process specifically involves: extracting the obtained reaction solution with dichloromethane and sodium bicarbonate solution or water, and then separating and purifying it using a silica gel column to obtain the hydrophobic alkyl monomer shown in Formula 5; the eluent used for column separation is petroleum ether:dichloromethane with a volume ratio of 1 to 100:

1.

9. The fluoropolymer nanodroplet according to claim 6, characterized in that: The molar ratio of the chain transfer agent QW, N,N-dimethacrylic acid monomer, and initiator is 1:(1-200):(0.1-0.5). The organic solvent is at least one of 1,4-dioxane, dimethyl sulfoxide, dimethylformamide, tetrahydrofuran, and toluene; Q is C6H5SS-, W is -CCNCH3CH2CH2COOH, R1 is -CH3, A is -O-, and when a=1, the chain transfer agent is 4-cyano-4-(thiobenzoyl)valerate, and the N,N-dimethylenoic acid monomer is ethyl 2-dimethylaminomethacrylate. The purification process specifically involves precipitating or ultrafiltration, dialysis, and lyophilizing the mixture after the reaction using one or more solutions selected from petroleum ether, diethyl ether, cyclohexane, methyl tert-butyl ether, and ethyl acetate.

10. The method for preparing fluoropolymer nanodroplets as described in claim 1, characterized in that: The preparation method of the fluoropolymer nanodroplets includes the following steps: Perfluorinated carbon, fluoropolymer, and photosensitizer are dissolved in an organic solvent and then sonicated. After the mixture is sonicated and evenly dispersed, an aqueous solution of polyvinyl alcohol is added and the mixture is sonicated again until it becomes an emulsion. The organic solvent is then removed by rotary evaporation or aeration stirring. Finally, dialysis is performed to remove unloaded small molecules, thus obtaining fluoropolymer nanodroplets. The organic solvent is at least one of dichloromethane, tetrahydrofuran, and trichloromethane; The ultrasonic power is 50-1500W; The mass concentration of the polyvinyl alcohol aqueous solution is 0.5% to 5%; The volume ratio of the organic solvent to the polyvinyl alcohol aqueous solution is 1:5-20; the ratio of the amount of perfluorinated carbon, fluoropolymer, and photosensitizer is 0.01-1 mL:0.5-10 mg:0.1-5 mg. The dialysis used a molecular weight cutoff of 1~10kDa.

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