Mesoporous polydopamine-based drug delivery system as well as preparation method and application thereof
By loading hydrophobic drugs and hydrophilic enzymes on mesoporous polydopamine carriers and combining them with hyaluronic acid coating and manganese acetate mesoporous polydopamine drug delivery systems, the delivery problems of hydrophobic drugs and enzymes are solved, efficient tumor targeted therapy is achieved, the limitations of traditional drug delivery are overcome, and the therapeutic effect and safety are improved.
Patent Information
- Application Number
- CN202510598964.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-10
- Publication Date
- 2025-09-12
AI Technical Summary
In the existing technology, hydrophobic drugs such as simvastatin have low solubility in aqueous solution and are difficult to deliver effectively, and hydrophilic enzymes such as cholesterol oxidase have poor stability in the body and are difficult to achieve efficient synergistic effects, leading to limitations of traditional chemotherapy and enzyme therapy.
Mesoporous polydopamine (mPDA) is used as a carrier to load the hydrophobic drug simvastatin and the hydrophilic enzyme cholesterol oxidase, and is coated with hyaluronic acid to achieve targeted release of the drug and stability of the enzyme. The interaction between the mesoporous structure and the surfactant groups is utilized to combine manganese acetate to participate in the redox reaction, thereby enhancing the killing effect of tumor cells.
It achieves efficient loading of hydrophobic drugs and stability of enzymes, increases the enrichment concentration of drugs in tumor tissues, reduces toxic side effects on normal tissues, and significantly improves the anti-cancer effect through multi-target synergistic treatment.
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Figure CN120617539A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of anti-tumor drug preparation, and more specifically, to a drug delivery system based on mesoporous polydopamine, a preparation method and an application thereof. Background Art
[0002] In the field of cancer treatment, developing efficient and safe treatment strategies has always been the core goal of research. In traditional chemotherapy methods, the delivery of hydrophobic drugs faces many severe challenges. On the one hand, hydrophobic drugs have extremely low solubility in aqueous solutions, making it difficult for the drugs to effectively dissolve and enter the blood circulation system, and unable to fully exert their therapeutic efficacy. On the other hand, it is difficult to accurately deliver hydrophobic drugs to the tumor site. The drugs are easily hindered by various physiological barriers during transportation in the body, making it difficult to reach effective therapeutic concentrations in tumor tissues, and may also produce unnecessary toxic side effects on normal tissues.
[0003] In the field of drug delivery, efficiently delivering drugs of varying properties to target sites is a key challenge. Hydrophobic drugs, such as simvastatin (Si), have low solubility in aqueous solutions, making them difficult to deliver effectively, severely limiting their clinical applications. Hydrophilic enzymes, such as cholesterol oxidase (CHOX), are less stable in the body and are easily inactivated by various factors, thus reducing their therapeutic efficacy.
[0004] In recent years, enzyme therapy, as an emerging cancer treatment strategy, has demonstrated unique advantages. For example, enzymes can specifically act on certain key metabolic pathways within tumor cells, thereby achieving targeted killing of tumor cells. Furthermore, compared to traditional chemotherapy drugs, enzymes have relatively fewer toxic side effects. However, single enzyme therapy also has limitations. For example, enzymes are relatively unstable and are easily degraded by proteases in the body, resulting in reduced activity and affecting therapeutic efficacy.
[0005] Polydopamine is a polymer with good biocompatibility, adhesion and chemical reactivity. Mesoporous polydopamine (mPDA), as a new type of mesoporous material, not only has a high specific surface area and a regular and ordered pore structure, but also retains the excellent properties of polydopamine. These properties make mesoporous polydopamine show great potential in the field of drug delivery, and its rich pore structure can provide space for drug loading. The high specific surface area of mPDA provides a large number of adsorption sites for the loading of drugs and enzymes, and the ordered pore structure is conducive to the storage and slow release of drugs and enzymes. In addition, polydopamine itself has good biocompatibility, which can reduce the immunogenicity of nanomaterials in the body and improve their safety. Therefore, the cancer treatment strategy based on the simultaneous loading of hydrophobic drugs and enzymes on mPDA is expected to overcome the shortcomings of traditional hydrophobic drug delivery and open up new avenues for cancer treatment. Summary of the Invention
[0006] The present invention provides a drug delivery system based on mesoporous polydopamine, a preparation method and an application thereof, in order to solve the problems in the prior art that hydrophobic drugs and hydrophilic enzymes are difficult to be effectively delivered and difficult for the two to act synergistically.
[0007] According to one aspect of the present invention, a drug delivery system based on mesoporous polydopamine is provided, characterized in that it includes a mesoporous polydopamine carrier, the pores of the mesoporous polydopamine carrier are loaded with a hydrophobic drug, a hydrophilic enzyme and manganese acetate, the hydrophobic drug is simvastatin, and the hydrophilic enzyme is cholesterol oxidase.
[0008] Preferably, based on the above scheme, the structural formula of the drug delivery system is: mPDA-SC-Mn-HA.
[0009] The present invention provides a method for preparing a drug delivery system based on mesoporous polydopamine, comprising the following steps:
[0010] Step 1: dopamine hydrochloride (DA·HCl) and F127 are added to a mixed solution of water and ethanol in a volume ratio of 1:1 and stirred. 1,3,5-trimethylbenzene is added and stirred for 30 minutes. Then, 5 mL of ammonia water is added and stirred for 12 hours. After the reaction is completed, the template is ultrasonically removed using a mixed solution of acetone and ethanol in a volume ratio of 1:1. The mPDA carrier is washed with ultrapure water to obtain a mesoporous polydopamine (mPDA) carrier.
[0011] Step 2: Add simvastatin, cholesterol oxidase, and manganese acetate to the mesoporous polydopamine dispersion solution. After stirring at room temperature for 12 hours, add hyaluronic acid (HA) solution dropwise using a constant pressure separatory funnel and stir at room temperature for 24 hours. After polymerization, mPDA-SC-Mn-HA is obtained.
[0012] Preferably, the simvastatin, cholesterol oxidase, manganese ion (Mn 2+ The mass ratio of ) to mesoporous polydopamine is 1:1.
[0013] Preferably, based on the above scheme, in step 2, the hydrophobic drug simvastatin is dispersed in a small amount of ethanol solution, and then added to the mesoporous polydopamine solution and stirred for adsorption loading.
[0014] Preferably, based on the above scheme, in step 2, the loading of cholesterol oxidase is achieved by utilizing the interaction between the active groups on the surface of the mesoporous polydopamine carrier and the enzyme.
[0015] Preferably, based on the above scheme, in step 2, hyaluronic acid is used to coat the mesoporous polydopamine loaded with simvastatin, cholesterol oxidase and manganese acetate.
[0016] Preferably, based on the above scheme, in step 2, when hyaluronic acid specifically binds to the CD44 receptor on the surface of tumor cells, the drug will not leak during blood circulation and will be concentrated in the tumor site.
[0017] Mesoporous polydopamine-based drug delivery system is applied to inhibit tumor cell proliferation.
[0018] Preferably, based on the above scheme, the cholesterol oxidase oxidizes cholesterol that synthesizes cell membranes and maintains intracellular signal transduction, so as to destroy the cell membrane structure and function of tumor cells and inhibit the proliferation of tumor cells.
[0019] A drug delivery system based on mesoporous polydopamine is applied to inhibit tumor cell proliferation. 2+ Reactive oxygen species (ROS) are generated in tumor cells through Fenton-like reactions, leading to cell membrane damage, protein inactivation and DNA fragmentation.
[0020] The present invention discloses a mesoporous polydopamine-based drug delivery system, a preparation method, and an application thereof. The present invention uses mesoporous polydopamine and a substrate to simultaneously load hydrophobic simvastatin, hydrophilic cholesterol oxidase, and manganese acetate, and a nanocomposite material coated with hyaluronic acid. The nanocomposite material has good drug loading capacity, realizes targeted drug release, kills tumor cells, and inhibits their proliferation by consuming the cholesterol in tumor cells through the drug.
[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0022] Mesoporous polydopamine has a highly ordered mesoporous structure and a huge specific surface area, providing abundant loading sites for the hydrophobic drug simvastatin, the enzyme cholesterol oxidase, and the metal compound manganese acetate. As a hydrophobic statin, simvastatin has extremely low solubility in aqueous solution, making it difficult to achieve efficient delivery. The mesoporous structure of mPDA can stably encapsulate simvastatin in the pores through physical adsorption and hydrophobic interactions, significantly increasing its loading capacity and solving the problem of low loading efficiency of hydrophobic drugs by traditional carriers. For cholesterol oxidase and manganese acetate, the surface and pores of mPDA can also provide a suitable microenvironment to ensure the activity of the enzyme and the stability of the metal compound, achieving efficient co-loading of multiple drugs and laying the foundation for synergistic treatment.
[0023] Simvastatin can interfere with the cholesterol metabolism of tumor cells and induce apoptosis of tumor cells by inhibiting the mevalonate pathway. Cholesterol oxidase can specifically catalyze the oxidation of cholesterol, consume cholesterol in tumor cells, destroy the integrity of cell membranes, and affect the growth and proliferation of tumor cells. Manganese acetate, as a metal compound, can participate in redox reactions in the tumor microenvironment, enhance the production of reactive oxygen species (ROS), and further induce tumor cell death. The three, in synergy with the mPDA carrier, can exert a multi-target, multi-pathway combined anti-cancer effect, produce significant synergistic therapeutic effects, overcome the limitations of single drug treatment, further improve the therapeutic effect of drugs, and ultimately realize a combined treatment model combining chemotherapy and enzyme therapy.
[0024] Hyaluronic acid (HA) can specifically bind to the CD44 receptor that is usually overexpressed on the surface of tumor cells. This enables the HA-coated MPDA drug delivery system to actively target tumor cells, increase the drug's enrichment concentration in tumor tissue, and reduce its distribution in normal tissues, thereby reducing the drug's toxic side effects on normal cells and improving the safety and effectiveness of treatment. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.
[0026] In the picture:
[0027] Figure 1 This is a transmission electron microscopy image of mPDA-SC-Mn-HA in Example 1.
[0028] Figure 2 2 is the particle size and potential diagram of mPDA-SC-Mn-HA in Example 1.
[0029] Figure 3 This is the ultraviolet absorption spectrum of mPDA-SC-Mn-HA in Example 1.
[0030] Figure 4 2 is the drug release curve of mPDA-SC-Mn-HA under different conditions in Example 1.
[0031] Figure 5 This is a graph showing cholesterol consumption of mPDA-SC-Mn-HA under different conditions in Example 1.
[0032] Figure 6 This is a diagram showing the enzyme stability effect of mPDA-SC-Mn-HA in Example 1.
[0033] Figure 7 This is the TMB oxidation curve of mPDA-SC-Mn-HA in Example 1.
[0034] Figure 8 This is the MB degradation curve of mPDA-SC-Mn-HA in Example 1.
[0035] Figure 9 This is the ATP detection curve of mPDA-SC-Mn-HA in Example 1.
[0036] Figure 10 These are the cytotoxicity test results of mPDA-SC-Mn-HA in Example 1.
[0037] Figure 11 This is a schematic diagram of the synthesis principle of the preparation method of the mesoporous polydopamine-based drug delivery system of the present invention. DETAILED DESCRIPTION
[0038] The following embodiments of the present invention are described in further detail with reference to the accompanying drawings and examples. The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.
[0039] It will be understood that when used in this specification and the appended claims, the term "comprising" indicates the presence of described features, integers, steps, operations, elements and / or components, but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or collections.
[0040] It should be further understood that the term "and / or" used in this specification and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.
[0041] In addition, in the description of the present application, the terms "first", "second", etc. are only used to distinguish the description and cannot be understood as indicating or implying relative importance.
[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the specific embodiments of the present invention will be described below with reference to the accompanying drawings. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings and other embodiments can be obtained based on these drawings without inventive work.
[0043] The reagents used in the following examples are all commonly available commercial products. Unless otherwise specified, "water" refers to "deionized water." The PBS buffer used in the following examples has a concentration of 10 mM unless otherwise specified.
[0044] See also Figure 11 As shown, the preparation method of a drug delivery system based on mesoporous polydopamine of the present invention comprises the following steps:
[0045] (1) Synthesis of mPDA
[0046] Dopamine hydrochloride (DA·HCl) and F127 were added to a 1:1 water / ethanol mixture with stirring. 1,3,5-Trimethylbenzene was added and stirring continued for 30 minutes. Then, 5 mL of ammonia was added and stirred for 12 hours. After the reaction, the template was ultrasonically removed using a 1:1 acetone / ethanol mixture, and the mPDA support was washed with ultrapure water to obtain the resultant product.
[0047] (2) Simvastatin was dissolved in a small amount of ethanol solution and then added dropwise to the mPDA mixed solution, stirred at room temperature for 12 h, centrifuged and washed, and the precipitate was collected to obtain mPDA-Si nanoparticles. Cholesterol oxidase and manganese acetate were added to the mPDA-Si dispersed solution, stirred at room temperature for 12 h, centrifuged and washed, and the precipitate was collected to obtain mPDA-SC-Mn nanoparticles.
[0048] (3) Hyaluronic acid (HA) solution was added dropwise using a constant pressure separatory funnel and stirred at room temperature for 24 h. After polymerization was completed, the precipitate was collected by centrifugation to obtain mPDA-SC-Mn-HA.
[0049] in, Figure 11 This is a schematic diagram of the preparation method of the mesoporous polydopamine-based drug delivery system of the present invention.
[0050] The present invention also provides the application of the drug delivery system based on mesoporous polydopamine simultaneously loaded with hydrophobic drugs and hydrophilic enzymes in the field of anti-cancer.
[0051] This application designs and constructs a new type of biomimetic drug delivery system that combines hydrophobic drug loading and targeted delivery.
[0052] In order to further illustrate the effects of the present invention, specific embodiments will be used to illustrate the present invention.
[0053] Example 1 A method for preparing a drug delivery system based on mesoporous polydopamine that simultaneously loads hydrophobic drugs and hydrophilic enzymes, the specific steps are as follows:
[0054] 1g of triblock polymer Pluronic F127 and 0.5g of dopamine hydrochloride were added to a 100mL mixture of ethanol and water (1:1 volume ratio). 2mL of 1,3,5-trimethylbenzene was then added. After stirring at room temperature for 30 minutes, 5mL of ammonia was added and stirring continued at room temperature for 12 hours. Nanoparticles were collected by centrifugation, and the template was removed by sonication with a mixture of acetone and ethanol for 30 minutes. The nanoparticles were then washed three times with ultrapure water to obtain mPDA.
[0055] 10 mg of mesoporous polydopamine was dispersed in water, and 10 mg of simvastatin dissolved in ethanol solution was added. After stirring at room temperature for 12 h, the unadsorbed simvastatin was removed by centrifugation and the precipitate was collected to obtain mPDA-Si nanoparticles.
[0056] The collected mPDA-Si precipitate was dispersed in water, and 10 mg cholesterol oxidase and 45 mg manganese acetate (10 mg Mn 2+ ), stirred at room temperature for 12 h and then centrifuged to remove the adsorbed cholesterol oxidase and Mn 2+ The precipitate was collected to obtain mPDA-SC-Mn nanoparticles.
[0057] The collected mPDA-SC-Mn nanoparticles were dispersed in 10 mL of water, and 4 mg mL -1 The hyaluronic acid solution was stirred at low speed at room temperature for 24 hours, and the uncoated hyaluronic acid was removed by centrifugation. The precipitate was collected to obtain mPDA-SC-Mn-HA nanoparticles.
[0058] In order to observe the morphology of mPDA-SC-Mn-HA in detail, the mPDA-SC-Mn-HA nanoparticles obtained in Example 1 were observed under a transmission electron microscope, wherein, Figure 1 This is the material morphology observed by transmission electron microscopy. It can be seen that the surface of mPDA is covered with a lighter-colored HA shell with a size of about 106 nm, and the successful loading of Mn atoms can be clearly seen in mPDA-SC-Mn-HA.
[0059] In order to measure the particle size of the material in PBS, mPDA-SC-Mn-HA was dispersed in a PBS solution with a pH of 7.4. Figure 2 As shown in the figure, the particle size of mPDA was 244.3±73.83 nm and the particle size of mPDA-SC-Mn-HA was 307.85±99.49 nm as detected by a laser particle size analyzer. The increase in particle size indicated the effective coating of hyaluronic acid.
[0060] The potential of mPDA is -42.97±1.41 nm, and the potential of mPDA-SC-Mn loaded with simvastatin, cholesterol oxidase and manganese acetate is -19.23±0.81 mV. The increase in potential may be due to the fact that although simvastatin is a neutral molecule and does not directly contribute to the charge, its hydrophobicity may reduce the exposure of the hydrophilic carboxyl groups on the PDA surface, weakening the negative charge; Mn 2+ Adsorbed onto the negatively charged PDA surface, the negative charge is neutralized by electrostatic interaction; although the surface of cholesterol oxidase is negatively charged, its load can cover the negatively charged groups on the PDA surface to reduce the exposure of negative charge, and its negatively charged residues may react with Mn 2+ Combined with enzyme-manganese complex, masking the negative charge of the enzyme itself, and even introducing Mn 2+ The positive contribution of multiple effects leads to an increase in the charge of mPDA-SC-Mn. After loading with hyaluronic acid, the potential of mPDA-SC-Mn-HA is -46.23±1.81mV. Since hyaluronic acid is a linear polysaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine, its molecular structure contains a large number of carboxyl groups (-COOH). Under physiological pH conditions (about 7.4), the carboxyl groups will dissociate and release hydrogen ions (H + ), which gives the hyaluronic acid molecules a negative charge. Therefore, the change in potential also confirms the loading of simvastatin, cholesterol oxidase, and manganese acetate, as well as the coating of the hyaluronic acid shell.
[0061] like Figure 3 Figure 2 shows the UV absorption spectra of mPDA-SC-Mn-HA of the present invention. The UV absorption spectra of mPDA-SC-Mn-HA at different concentrations show that the material absorbs UV light at 808 nm, and the absorption increases with increasing concentration. mPDA and mPDA-SC-Mn-HA also exhibit similar broad-spectrum absorption properties.
[0062] In order to investigate the in vitro drug release performance of mPDA-SC-Mn-HA, the present invention used the BCA method to determine the in vitro drug release of cholesterol oxidase (CHOX), and used ICP to determine the release of Mn 2+ The in vitro drug release of Si was determined by high performance liquid chromatography (HPLC).
[0063] Si, CHOX, Mn in mPDA-SC-Mn-HA 2+ Calculation of drug loading and encapsulation efficiency: After HA coating, all the supernatants obtained by washing with water and centrifugation were used to measure the drug loading capacity DLC and encapsulation efficiency DLE. The specific calculation formulas are shown in the following formulas (1) and (2). The cholesterol oxidase (CHOX) concentration in the supernatant was determined by BCA method, and Mn was determined by ICP. 2+The concentration of Si was determined by HPLC, and the concentration of Si, CHOX and Mn 2+ Standard curve determination of Si, CHOX and Mn in the supernatant 2+ The mass of drug loaded = the mass of drug delivered - the mass of APS in the supernatant.
[0064] DLC (wt.%) = (loaded drug mass / drug-loaded mPDA-SC-Mn-HA mass) × 100% Formula (1);
[0065] DLE (wt.%) = (loaded drug mass / delivered drug mass) × 100% Formula (2);
[0066] Composed of Si, CHOX and Mn 2+ The standard curve, the amount of each component and the mass of the final product can be used to calculate the encapsulation efficiency of Si in the supernatant to be 82% and the drug loading to be 15.53%; the encapsulation efficiency of CHOX to be 74.61% and the drug loading to be 17.27%; 2+ The encapsulation efficiency is 83.2% and the drug loading capacity is 21.63%; it can be seen that the prepared mPDA-SC-Mn-HA has a high encapsulation and loading performance for anticancer drug molecules.
[0067] In order to further test its in vitro drug release performance, the present invention took 1 mL of mPDA-SC-Mn-HA (1 mg / mL, prepared by pH = 7.4 PBS buffer) and added it to a dialysis bag (MWCO = 3500) with or without hyaluronidase. Subsequently, the dialysis bag was completely immersed in a test tube containing 10 mL of PBS solution (pH = 7.4), and the test tube was placed in a 37°C shaker for shaking. At predetermined time intervals, 3 mL of PBS external solution was taken out and 3 mL of the corresponding PBS buffer was added. The release amount of CHOX was determined by the BCA protein kit, and the Mn was determined by the ICP method. 2+ The amount of release.
[0068] The cumulative release of the drug is calculated as follows:
[0069]
[0070] E r Indicates the cumulative release of the drug, V e represents the replacement volume of PBS; V0 represents the total volume of release medium, C i represents the concentration of the released liquid during the ith replacement sampling; m drug represents the total mass of the drug carried by the nanoparticles; n represents the number of times PBS is replaced.
[0071] The test results are as follows Figure 4It was shown that in the absence of hyaluronidase, CHOX and Mn 2+ The cumulative release rate within 24 hours was low, not exceeding 20%. In the presence of hyaluronidase, CHOX and Mn 2+ The cumulative release rate within 24 hours can reach 80%.
[0072] To test the cholesterol consumption ability of mPDA-SC-Mn-HA, the present invention incubated PBS, 80 μg / mL CHOX, 100 μg / mL mPDA-Si-Mn-HA with HAase, and mPDA-SC-Mn-HA with HAase with 4 μM cholesterol at 37°C for 24 hours. The cholesterol content was determined using a cholesterol detection kit, and the peak value at 585 nm was detected by fluorescence to evaluate its cholesterol consumption ability.
[0073] The test results are as follows Figure 5 As shown, after HAase degraded the HA shell of mPDA-SC-Mn-HA, its cholesterol consumption capacity was comparable to that of pure CHOX. However, after HAase degraded the HA shell of mPDA-S-Mn-HA, its cholesterol consumption capacity was much lower than that of mPDA-SC-Mn-HA and pure CHOX. These experimental results indicate that the released drug from mPDA-SC-Mn-HA can effectively consume cholesterol.
[0074] Furthermore, in order to test the enzyme stability of mPDA-SC-Mn-HA, the present invention incubated PDA-Si-CHOX-Mn-HA (100 μg / mL) with cholesterol oxidase (CHOX, 80 μg / mL) at different temperatures (25, 37, 50, 60 and 70°C) for 2 hours and at different pH conditions (4, 5, 6, 7, 8 and 9) for 48 hours. Subsequently, 80 μg / mL cholesterol was added to the mixed system and incubated for another hour. The enzyme activity was determined using a cholesterol detection kit. The enzyme activity of PDA-Si-CHOX-Mn-HA and free CHOX at 45°C and pH = 5 was defined as 100% for relative activity calculation.
[0075] The results are as follows Figure 6 As shown in the figure, the enzyme stability of mPDA-SC-Mn-HA is similar to that of CHOX. It is relatively stable at low temperatures, but the enzyme will be inactivated at high temperatures, resulting in reduced stability. In addition, the enzyme stability decreases with increasing pH, indicating that the enzyme has good stability under weakly acidic conditions.
[0076] In order to test the ability of mPDA-SC-Mn-HA to produce hydroxyl radicals, the cholesterol oxidase in mPDA-SC-Mn-HA was reacted with cholesterol to generate hydrogen peroxide (H2O2). 2+ Hydroxyl radicals (·OH) are generated through a Fenton-like reaction under the catalysis of .
[0077] (1) TMB oxidation reaction
[0078] 3,3',5,5'-Tetramethylbenzidine (TMB) can be oxidized by ·OH and turned from colorless to blue. The color reaction of 3,3',5,5'-Tetramethylbenzidine (TMB) was used to detect the ability of mPDA-SC-Mn-HA to produce ·OH. -1 mPDA-SC-Mn-HA was mixed with 0, 10, 20, and 40 μg of cholesterol for 30 min, followed by the addition of TMB solution. Finally, a UV spectrophotometer was used to scan the UV absorption spectrum in the range of 200-1000 nm, and the absorbance at 650 nm was measured and photographed. The results are shown in Figure 2. Figure 7 As shown in the figure, with the increase of concentration, the peak value of TMB at 650 nm gradually increased, indicating that mPDA-SC-Mn-HA can produce ·OH to oxidize TMB.
[0079] (2) MB degradation experiment
[0080] Methylene blue (MB) can be degraded by ·OH and changes from blue to colorless or light yellow. The ability of mPDA-SC-Mn-HA to produce ·OH was detected by methylene blue colorimetric reaction. -1 mPDA-SC-Mn-HA was mixed with 0, 10, 20, and 40 μg of cholesterol for 30 min, and then MB solution was added. The absorption spectrum in the range of 200-1000 nm was scanned using a UV spectrophotometer, and the absorbance at 617 nm was measured and photographed. The results are shown in Figure 2. Figure 8 As shown in the figure, with the increase of concentration, the peak value of MB at 617 nm gradually decreased, indicating that mPDA-SC-Mn-HA can produce ·OH to degrade MB.
[0081] (3) APF probe detects the ability of materials to generate ·OH
[0082] 200 μL of 100 μg mL -1 mPDA-SC-Mn-HA was mixed with 10 μM cholesterol, and APF probe was randomly added. The changes in fluorescence intensity at 515 nm were measured over time using a fluorescence spectrophotometer. Figure 9As shown in the figure, the peak value of APF at 515 nm gradually increased with the passage of time, indicating that mPDA-SC-Mn-HA can maintain good ·OH generation performance over time.
[0083] TMB, MB, and APF all showed that mPDA-SC-Mn-HA had a good and long-lasting ability to generate ·OH, which may be due to the fact that cholesterol oxidase oxidized cholesterol to generate H2O2, while Mn 2+ It can undergo a Fenton-like reaction with H2O2 in cancer cells and newly generated H2O2 to produce ·OH.
[0084] To investigate the cytotoxicity of mPDA-SC-Mn-HA, the present invention used frozen tumor cells (4T1 cells) from the inventor's laboratory. The cell culture medium used was commercially available DMEM medium containing 10% fetal bovine serum albumin and 1% penicillin-streptomycin (hereinafter referred to as DMEM complete medium). All cells were incubated in the cell culture medium in a cell culture incubator containing 5% CO2 at 37°C.
[0085] To investigate the cytotoxicity of mPDA-SC-Mn-HA, the present invention used a CCK-8 assay to test the cytotoxicity of mPDA-SC-Mn-HA against tumor cells (4T1 cells). The CCK-8 assay is a rapid, highly sensitive assay widely used for cell proliferation and cytotoxicity. It can be reduced to produce a yellow, water-soluble formazan. Greater and faster cell proliferation results in a darker color; greater cytotoxicity results in a lighter color.
[0086] The detection method is as follows: Mouse breast cancer cells (4T1) were seeded at a density of 5×103 cells / well in 100 μL of DMEM complete medium containing 10% FBS using a 96-well plate. After the 96-well plate was placed in a 37°C incubator (5% CO2) and cultured for 24 hours, 100 μL of a series of APS@TRP / PDA DMEM complete medium solutions with a concentration gradient were added to each column (the concentration was the same in the same column). After the cells were placed in a 37°C incubator (5% CO2) and incubated in the dark for 24 hours, 20 μL of CCK-8 solution was added to each well (try to avoid bubbles during the addition process) and incubated in a 37°C incubator (5% CO2) for another 4 hours. The absorbance (OD) at 450 nm was measured using a microplate reader to calculate the relative cell viability. The OD value in the cytotoxicity experiment was the average of 6 independent parallel samples, and the results were expressed as the mean ± standard deviation (SD). The relative cell viability is usually calculated according to formula (3):
[0087] Viability(%)=(ODsample / ODcontrol)×100 Formula (3);
[0088] Among them, ODcontrol is the cell absorbance value detected before the addition of APS@TRP / PDA, and ODsample is the cell absorbance value detected after the addition of APS@TRP / PDA material.
[0089] In addition, CCK-8 kit was used to detect
[0090] Group ①mPDA-C-Mn-HA,
[0091] Group ②mPDA-S-Mn-HA,
[0092] Group ③mPDA-SC-HA,
[0093] Group ④mPDA-SC-Mn-HA,
[0094] Test results such as Figure 9 As shown in FIG, mouse breast cancer cells (4T1) were used as experimental subjects, and the horizontal axis was the mPDA concentration in mPDA-SC-Mn-HA materials with different concentrations (calculated according to the standard curve of PDA absorbance at 808 nm). Figure 10 As shown in the figure, the experimental results show that at low concentrations, the killing effect of mPDA-C-Mn-HA on 4T1 cells may be due to the fact that cholesterol oxidase can consume cholesterol in tumor cells, reduce the intracellular cholesterol level, affect the fluidity and stability of the cell membrane, and then interfere with the normal metabolism and signal transduction of tumor cells, leading to tumor cell cycle arrest and thus inhibiting tumor cell proliferation. 2+ It can react with hydrogen peroxide (H2O2) generated by cholesterol oxidation by cholesterol oxidase to produce hydroxyl radicals (·OH) and other highly oxidative ROS. These ROS can attack biomacromolecules in tumor cells, such as lipids, proteins and DNA, leading to cell membrane damage, protein inactivation and DNA breakage, thereby inducing tumor cell apoptosis. At low concentrations, the survival rate of 4T1 cells in the mPDA-S-Mn-HA, mPDA-SC-HA and mPDA-SC-Mn-HA groups was only about 50%. This may be because the rapid proliferation of tumor cells requires a large amount of cholesterol to synthesize cell membranes and maintain intracellular signal transduction. Simvastatin can reduce total cholesterol levels and thus inhibit the growth of tumor cells. Simvastatin can also react with cholesterol oxidase and Mn 2+The results showed that mPDA-SC-Mn-HA has a good synergistic therapeutic effect. At higher concentrations, the survival rate of 4T1 cells in the mPDA-SC-Mn-HA group was less than 20%. These experimental results show that mPDA-SC-Mn-HA can significantly inhibit cell proliferation and induce cell apoptosis.
[0095] Finally, the method of this application is only a preferred embodiment and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.
Claims
1. A drug delivery system based on mesoporous polydopamine, characterized in that: The invention comprises a mesoporous polydopamine carrier, wherein the pores of the mesoporous polydopamine carrier are loaded with hydrophobic drugs, hydrophilic enzymes and manganese acetate, wherein the hydrophobic drugs are simvastatin (Si), and the hydrophilic enzymes are cholesterol oxidase (CHOX).
2. The drug delivery system based on mesoporous polydopamine according to claim 1, characterized in that The structural formula of the drug delivery system is: mPDA-SC-Mn-HA.
3. A method for preparing a drug delivery system based on mesoporous polydopamine, characterized in that: The following steps are included: Step 1: dopamine hydrochloride (DA·HCl) and F127 are added to a mixed solution of water and ethanol in a volume ratio of 1:1 and stirred. 1,3,5-trimethylbenzene is added and stirred for 30 minutes. Then, 5 mL of ammonia water is added and stirred for 12 hours. After the reaction is completed, the template is ultrasonically removed using a mixed solution of acetone and ethanol in a volume ratio of 1:
1. The mPDA carrier is washed with ultrapure water to obtain a mesoporous polydopamine (mPDA) carrier. Step 2: Add simvastatin, cholesterol oxidase, and manganese acetate to the mesoporous polydopamine dispersion solution. After stirring at room temperature for 12 hours, add hyaluronic acid (HA) solution dropwise using a constant pressure separatory funnel and stir at room temperature for 24 hours. After polymerization, mPDA-SC-Mn-HA is obtained.
4. The method for preparing a drug delivery system based on mesoporous polydopamine according to claim 3, wherein: In the step 2, the hydrophobic drug simvastatin is dispersed in a small amount of ethanol solution, and then added into the mesoporous polydopamine solution and stirred for adsorption loading.
5. The method for preparing a drug delivery system based on mesoporous polydopamine according to claim 3, wherein: In step 2, the cholesterol oxidase is loaded by utilizing the interaction between the active groups on the surface of the mesoporous polydopamine carrier and the enzyme.
6. The method for preparing a drug delivery system based on mesoporous polydopamine according to claim 3, wherein: In the step 2, hyaluronic acid is used to coat the mesoporous polydopamine loaded with simvastatin, cholesterol oxidase and manganese acetate.
7. The method for preparing a drug delivery system based on mesoporous polydopamine according to claim 3, wherein: In step 2, when hyaluronic acid specifically binds to the CD44 receptor on the surface of tumor cells, the drug is prevented from leaking during blood circulation and is concentrated in the tumor site.
8. Mesoporous polydopamine-based drug delivery systems are used to inhibit tumor cell proliferation and induce tumor cell apoptosis.
9. The use of a drug delivery system based on mesoporous polydopamine as claimed in claim 8, characterized in that: The cholesterol oxidase oxidizes cholesterol that synthesizes cell membranes and maintains intracellular signal transduction, thereby destroying the cell membrane structure and function of tumor cells and inhibiting the proliferation of tumor cells.
10. The drug delivery system based on mesoporous polydopamine as claimed in claim 8 is used to inhibit tumor cell proliferation, characterized in that: Mn in the drug delivery system 2+ Reactive oxygen species (ROS) are generated in tumor cells through Fenton-like reactions, leading to cell membrane damage, protein inactivation and DNA fragmentation.