Preparation method of pH-responsive polydopamine coating loaded dexamethasone and BMP-2 mesoporous silicon nanobone system

Abstract

The application belongs to the field of medicine and provides a preparation method of a pH-responsive polydopamine coating loaded dexamethasone and BMP-2 mesoporous silica nanobone forming system. The application coats a layer of polydopamine coating on the dexamethasone loaded mesoporous silica nanoparticles, and then loads BMP-2. The polydopamine coating serves as a pH-responsive mesoporous blocking material to control drug release and as a functional platform to load growth factors, realizes chemical / physical connection of the protein BMP-2, and thus constructs a 'one-body double-drug' mesoporous silica drug loading system, effectively improving the drug loading rate and the controlled release capacity.

Description

Technical Field

[0001] This invention belongs to the field of medicine, and specifically relates to a method for preparing a pH-responsive polydopamine-coated dexamethasone and BMP-2 mesoporous silica nano-osteogenic system. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] The rapid development of bone tissue engineering has brought opportunities for bone defect repair. Bone tissue engineering involves three important factors: tissue engineering scaffolds, cell growth factors, and seed cells. Among numerous osteogenic growth factors, bone morphogenetic protein-2 (BMP-2) is the most widely used and has the best osteogenic effect. In clinical practice, to achieve long-term effective treatment, the dosage of BMP-2 used is much higher than the normal physiological content in natural bone. High-dose use of BMP-2 can cause many side effects, such as tissue swelling, inflammatory response, ectopic osteoogenesis, and tumor induction. Studies have confirmed that vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet-rich plasma (PRP) can promote the osteogenic effect of BMP-2, and their combined use has a synergistic effect. Compared with growth factors that are complex to prepare, expensive, and easily inactivated, dexamethasone can also enhance the osteogenic effect of BMP-2, is more readily available, and has a lower cost, playing a key role in inducing osteogenic differentiation of cells.

[0004] The osteogenic effect of BMP-2 is closely related to its dosage, but maintaining a sustained release at a certain concentration is more important. Therefore, the key to leveraging the synergistic effect of BMP-2 lies in developing a drug delivery system (DDS) that can simultaneously load BMP-2 and DEX. In numerous studies, mesoporous silicon nanoparticles (MSNs) have attracted widespread attention as an emerging drug carrier. MSNs possess significant advantages such as controllable particle size, tunable pores, easily modifiable surfaces, good biocompatibility, and stability. Their mesoporous structure can load small molecule drugs, and the surface of the nanoparticles is rich in silanol groups, allowing for the loading of various biomolecules such as genes, peptides, and proteins after functionalization. Furthermore, various mesoporous silicon drug delivery systems adapted to specific conditions are constantly being developed. For example, under endogenous or exogenous stimuli such as pH, enzymes, light, ultrasound, and magnetism, drugs can be released in a controlled or targeted manner, avoiding "burst release" while endowing MSNs with superior properties such as conditional responsiveness or targeting.

[0005] The emergence of polydopamine (PDA) coatings, a biomimetic material based on mussels, has provided a new approach for immobilizing biomolecules on nanoparticles. Even in very humid environments, mussels can still form strong bonds with various interfaces. Studies have found that mussel foot proteins contain a large number of lysine residues, 3,4-dihydroxy-L-phenylalanine (DOPA) residues, and hydroxyproline residues, similar to the structure of dopamine (DA). Inspired by this, researchers have synthesized polydopamine (PDA) coatings using the principle of spontaneous polymerization of dopamine under alkaline conditions, and these coatings have been widely applied in drug delivery, tissue engineering, and biosensing. Furthermore, studies have found that PDA exhibits some pH sensitivity, with accelerated degradation under acidic conditions, making it a promising candidate as a pH-responsive drug delivery platform.

[0006] In summary, existing research techniques have the following drawbacks:

[0007] (1) Although growth factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet-rich plasma (PRP) can promote the osteogenic effect of BMP-2, they are complicated to prepare, expensive, and easily inactivated. There is a lack of stable, economical, and easy-to-implement methods to reduce the dosage of BMP-2.

[0008] (2) Existing drug delivery systems mainly include polymer nanoparticles, inorganic nanoparticles, liposomes, etc., which are difficult to achieve drug delivery mode of multiple drugs. There is an urgent need for a "dual drug delivery" drug carrier.

[0009] (3) Existing drug delivery systems are mainly based on sustained release, and the controlled release effect is poor.

[0010] (4) At present, the surface modification of nanoparticles mainly involves grafting functional groups or molecules, which is complex, inefficient and unstable.

[0011] The paper "A Study on the Use of BMP-2 and Dexamethasone Loaded in a pH-Response Dual Drug-Loading System for Bone Repair" discloses a novel chitosan-mesoporous silica nanoparticle (chi-MSNs) carrier. However, the preparation process needs to be simplified, and the stability, drug loading rate, and controlled release capability still need to be improved. Summary of the Invention

[0012] To address the aforementioned issues, this invention provides a method for preparing a pH-responsive polydopamine-coated mesoporous silica nanoparticle osteogenic system loaded with dexamethasone and BMP-2. The invention first coats dexamethasone-loaded mesoporous silica nanoparticles with a polydopamine coating, then loads BMP-2. The polydopamine coating acts both as a pH-responsive mesoporous plug to regulate drug release and as a functional platform for loading growth factors, achieving chemical / physical linkage to the protein BMP-2. This constructs a "dual-drug" mesoporous silica drug-loading system, effectively improving drug loading rate and controlled release capability.

[0013] To achieve the above objectives, the present invention adopts the following technical solution:

[0014] A first aspect of the present invention provides a method for preparing a pH-responsive polydopamine-coated dexamethasone and BMP-2 mesoporous silica nano-osteogenic system, comprising:

[0015] Mesoporous silica nanoparticles were added to a dexamethasone ethanol solution, stirred for more than 24 hours in the dark, centrifuged, washed, and freeze-dried to obtain dexamethasone-loaded nanoparticles.

[0016] The dexamethasone-loaded nanoparticles were dispersed in a buffer solution, and then dopamine hydrochloride was added. The mixture was stirred in the dark for more than 12 hours, centrifuged, washed, and freeze-dried to obtain polydopamine-coated dexamethasone-loaded nanoparticles.

[0017] The polydopamine-coated dexamethasone nanoparticles were added to a BMP-2 solution, stirred for more than 4 hours, centrifuged, and freeze-dried to obtain polydopamine-coated dexamethasone and BMP-2 mesoporous silica nanoparticles.

[0018] The polydopamine PDA prepared by this invention is rich in various active groups on its surface, such as phenolic hydroxyl, quinone, amino and imine groups, which can undergo Michael addition or Schiff-base reaction with nucleophilic groups such as amino (-NH2) and thiol (-SH), and can be used as a platform for immobilizing biomacromolecules.

[0019] In some embodiments, the mass ratio of the mesoporous silica nanoparticles to dexamethasone is 100:6-10, or 100:8.

[0020] In some embodiments, the concentration of the dexamethasone ethanol solution is 2-6 mg / mL, or 4 mg / mL.

[0021] In some embodiments, the method for preparing the mesoporous silicon nanoparticles includes:

[0022] Step 1: Mix hexadecyltrimethylammonium bromide and sodium hydroxide with water until homogeneous, heat and stir continuously until completely dissolved. Then, add tetraethyl orthosilicate to react, centrifuge and wash to obtain the first product;

[0023] Step 2: Add the first product to an ethanol solution containing HCl, reflux the reaction, centrifuge, wash, and collect the second product;

[0024] Step 3: Repeat step 2 multiple times to remove the template agent hexadecyltrimethylammonium bromide. Then, centrifuge, wash, and dry to obtain mesoporous silica nanoparticles.

[0025] Currently, to address the issue of mesoporous silica's inability to directly load the protein BMP-2, surface modification methods are generally employed. However, this application's research has found that compared to modification with polymers such as chitosan and polylactic acid, polydopamine can achieve both controlled release of small-molecule dexamethasone with pH response and serve as a linking platform for macromolecules, enabling efficient linking of BMP-2 and achieving superior drug loading and sustained-release capabilities. Furthermore, the polydopamine coating formation process is simple, requiring no special solvents or stringent conditions, making it more convenient, stable, efficient, and solvent-free. Therefore, in some embodiments, the mass ratio of the dexamethasone-loaded nanoparticles to dopamine hydrochloride is 100:4-6, or 100:5.

[0026] In some embodiments, the mass concentration of the dexamethasone-loaded nanoparticles in the buffer solution is 1-4 mg / mL, or 2 mg / mL.

[0027] In some embodiments, the mass ratio of the polydopamine-coated dexamethasone nanoparticles to BMP-2 is 100:0.012-0.018, or 100:0.016.

[0028] In some embodiments, the concentration of the BMP-2 solution is 2-6 μg / mL, or 4 μg / mL.

[0029] In a second aspect, the present invention provides a pH-responsive polydopamine-coated dexamethasone and BMP-2 mesoporous silica nano-osteogenic system prepared by the above method.

[0030] A third aspect of the present invention provides the application of the above-described pH-responsive polydopamine-coated dexamethasone and BMP-2 mesoporous silica nano-osteogenic system in the pharmaceutical field.

[0031] Beneficial effects of the present invention

[0032] This method innovatively constructs a pH-responsive "integrated dual-drug" osteogenic system through a polydopamine coating, which can specifically address the above-mentioned shortcomings:

[0033] (1) Dexamethasone is widely used in clinical practice due to its anti-inflammatory, anti-allergic and immunosuppressive effects. It is low in cost, stable in nature and easy to obtain. By utilizing its osteogenic effect on BMP-2, the dosage of BMP-2 can be effectively reduced.

[0034] (2) Mesoporous silica has a special mesoporous structure. It can be used to load small molecule drugs such as dexamethasone. After surface functionalization, it can be used to load BMP-2 macromolecules, becoming a new carrier for multiple drug delivery.

[0035] (3) The reaction process of polydopamine (PDA) coating is simple and versatile. The surface has groups such as catechol, amine and imine, which can be used as a platform for macromolecular linkage by combining with ligands through supramolecular interactions such as electrostatics, van der Waals forces or hydrogen bonds.

[0036] (4) Polydopamine is pH responsive. At low pH, the degradation rate is accelerated, and the drug can be released accordingly, which is controllable.

[0037] (5) This invention uses low concentrations of dexamethasone to enhance the osteogenic effect of BMP-2 on MC3T3-E1 cells, providing a new theoretical basis for optimizing the use of growth factors in tissue engineering.

[0038] (6) The present invention prepared a pH-responsive polydopamine-coated mesoporous silica dual-drug-loaded nano-osteogene system, providing a new method for constructing conditionally responsive mesoporous silica multiple-drug-loaded modes.

[0039] (7) The present invention effectively reduces the dosage of BMP-2 through the synergistic effect of dexamethasone and BMP-2, which is of positive significance for reducing BMP-2 dose-related side effects and disease treatment costs. Attached Figure Description

[0040] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. Exemplary embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0041] Figure 1 A schematic diagram of a pH-responsive polydopamine-coated dexamethasone and BMP-2 mesoporous silica nano-osteogenetic system. Detailed Implementation

[0042] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0043] Terminology Explanation

[0044] BMPR refers to: BMP-2 receptor;

[0045] Endosome refers to: an intituent;

[0046] Lysosome refers to: lysosome;

[0047] RUNX 2 refers to Runt-related transcription factor-2.

[0048] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0049] Example 1

[0050] (1) Preparation of mesoporous silica nanoparticles

[0051] Weigh 1.0 g of hexadecyltrimethylammonium bromide (CTAB) and 0.28 g of sodium hydroxide (NaOH), and add them to 480 mL of deionized water. Heat the mixture to 80 °C and stir continuously until completely dissolved. Then, add 5 mL of tetraethyl orthosilicate (TEOS) dropwise to the solution and continue stirring for 2 hours. Finally, collect the product by centrifugation (8000 rpm, 10 min) and wash several times with water and ethanol. Disperse the sample in a 500 mL ethanol mixture containing 5 mL of HCl and reflux at 80 °C for 24 hours. Centrifuge and wash again to collect the sample, repeating this process three times to thoroughly remove the template agent CTAB. Mesoporous silica nanoparticles (MSNs) without template agent are obtained by centrifugation and washing, and then vacuum-dried to obtain MSNs powder samples.

[0052] (2) Preparation and drug loading detection of dexamethasone-loaded mesoporous silica nanoparticles

[0053] To construct a dexamethasone standard curve: Accurately weigh 30 mg of dexamethasone into a 100 mL volumetric flask, dissolve and dilute to the mark with ethanol, and mix well. Measure 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mL of this solution into 25 mL volumetric flasks, add ethanol to the mark, and mix well to obtain standard solutions with concentrations of 6, 12, 18, 24, 30, and 36 μg / mL, respectively. Measure the absorbance at 242 nm for each solution and construct a dexamethasone standard curve based on the results.

[0054] A 4 mg / mL dexamethasone ethanol solution was prepared. 100 mg of MSNs was added to 2 mL of the dexamethasone ethanol solution, stirred in the dark for 24 hours, centrifuged, and washed three times with deionized water. The resulting nanoparticles were then vacuum-dried using a freeze dryer to obtain dexamethasone-loaded DEX@MSNs. The washings and supernatant were collected together, and the absorbance at 242 nm was measured. The dexamethasone loading content (LC) was calculated using the DEX standard curve, as shown in the following formula:

[0055]

[0056] (3) Preparation and drug loading detection of polydopamine-coated dexamethasone mesoporous silica nanoparticles

[0057] 100 mg of DEX@MSNs were dispersed in 50 mL of Tris-HCl buffer (pH 8.5, 10 mM), and then 50 mg of dopamine hydrochloride (DA) was added. The mixture was stirred in the dark at room temperature for 12 hours to form a polydopamine (PDA) coating. The PDA-coated DEX@MSNs were then centrifuged, washed three times with deionized water to remove unpolymerized dopamine, and dried using a freeze dryer to obtain PDA-coated dexamethasone-loaded nanoparticles DEX@MSNs / PDA. The supernatant and washings were collected, and the absorbance at 242 nm was measured using a UV spectrophotometer. Finally, the drug loading of DEX was calculated using the above method.

[0058] (4) Preparation and drug loading detection of PDA-coated dexamethasone and BMP-2 mesoporous silica nanoparticles

[0059] Prepare 4 mL of a 4 μg / mL BMP-2 solution and add 100 mg of DEX@MSNs / PDA nanoparticles. Stir at 4 °C for 4 hours, centrifuge, and freeze-dry the product for 24 hours to obtain DEX@MSNs / PDA / BMP-2 nanoparticles simultaneously loaded with dexamethasone and BMP-2. Collect the supernatant and determine the BMP-2 concentration using a BMP-2 ELISA kit to calculate the drug loading.

[0060] (5) Characterization of nanoparticles

[0061] ① Scanning electron microscope (SEM)

[0062] The surface morphology and particle size of the nanoparticles were observed using SEM (ZEISS Sigma 300, Germany). A small sample was directly adhered to conductive adhesive and sputtered with gold for 45 seconds using a Quorum SC7620 sputtering system at a voltage of 10 mA. The sample was then examined using a scanning electron microscope with an accelerating voltage of 3 kV.

[0063] ② Transmission electron microscopy (TEM)

[0064] The morphology, structure, and particle size of the nanoparticles were observed using TEM (JEOL JEM-2100Plus, Japan). The sample was first dispersed in anhydrous ethanol and sonicated for 5 minutes to ensure uniform dispersion. The dispersion was then dropped onto a copper grid. After drying, the sample was loaded and images were captured at different magnifications.

[0065] ③ Particle size and zeta potential analysis

[0066] The particle size and zeta potential changes of the samples were detected using a nanoparticle size and zeta potential analyzer (DLS, Malvern Zetasizer Nano ZS90, UK).

[0067] ④ Specific surface area and pore size analysis

[0068] N2 adsorption-desorption tests were performed on the samples using a fully automated specific surface area and porosity analyzer (Micromeritics ASAP 2460, USA). Specific surface area and pore size distribution were calculated according to the Barret-Joyner-Halenda (BJH) and Brunauer-Emmett-Teller (BET) methods.

[0069] ⑤ Fourier Transform Infrared Spectroscopy (FTIR)

[0070] The chemical signature groups of the nanoparticles were analyzed using FTIR (Thermo Scientific Nicolet iS20, USA). After drying, a small amount of sample powder was placed directly on the fixed stage of the instrument for testing. The scanning range was 4000-400 cm⁻¹, and the resolution was 4 cm⁻¹.

[0071] ⑥ X-ray diffraction (XRD)

[0072] The mesoporous structure of nanoparticles was detected using XRD (Rigaku SmartLab SE, Japan). Diffraction peaks were detected in both the small-angle (2θ < 10°) and wide-angle scattering regions.

[0073] (6) Dexamethasone in vitro release

[0074] 20 mg of dexamethasone-loaded DEX@MSNs and DEX@MSNs / PDA nanoparticles were weighed out and added to 1 mL of PBS solution at different pH values ​​(pH = 7.4, 6.0, 5.0), then placed in dialysis bags with a molecular weight cutoff of 3500 Da. The dialysis bags were then placed in centrifuge tubes containing 4 mL of PBS at the same pH and shaken on a constant-temperature shaker (37℃, 100 rpm). At each detection time point, 2 mL of solution was taken out and 2 mL of PBS was added. Finally, the absorbance of the solutions taken out at each time point was measured at 242 nm using a UV spectrophotometer. The release amount of DEX was calculated based on the drug loading of DEX, and its release curve was plotted.

[0075] (7) BMP-2 in vitro release

[0076] Weigh 50 mg of DEX@MSNs / PDA / BMP-2 nanoparticles and place them in a 10 mL centrifuge tube. Add 5 mL of PBS buffer (pH = 7.4, 6.0, 5.0) and vortex in a constant temperature shaker (37℃, 80 rpm). Centrifuge at predetermined times and collect the supernatant for analysis. Determine the BMP-2 concentration according to the BMP-2 ELISA kit instructions. Set the absorption wavelength to 450 nm and measure the BMP-2 concentration in the supernatant using a microplate reader. Calculate the BMP-2 release amount and plot its release curve.

[0077] Comparative Example 1

[0078] Step 2 prepared the loaded dexamethasone mesoporous silica nanoparticles DEX@MSNs.

[0079] Comparative Example 2

[0080] The difference from Example 1 is that chitosan is used instead of dopamine hydrochloride. Specific steps include:

[0081] (1) Add 50 mg of chitosan to 10 mL of acetic acid solution with a mass concentration of 3% and stir magnetically for 12 h to obtain chitosan solution;

[0082] (2) Disperse 100 mg of DEX@MSNs in 40 mL of anhydrous ethanol, adjust the pH with acetic acid to make the pH between 3.5 and 4.5, then add 200 μL of silane coupling agent, react at room temperature for 3 h, add the chitosan solution, react at room temperature for 24 h, centrifuge, wash and dry.

[0083] Table 1

[0084]

[0085]

[0086] A comparison of Example 1 and Comparative Example 1 shows that the introduction of the polydopamine coating effectively loaded dexamethasone and BMP-2 into a single unit, while significantly improving the controlled-release capacity of dexamethasone. A comparison of Example 1 and Comparative Example 2 shows that, compared to chitosan, the polydopamine coating resulted in a higher drug loading rate for dexamethasone and BMP-2, and also significantly improved the controlled-release capacity for both.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a pH-responsive polydopamine-coated dexamethasone and BMP-2 mesoporous silica nano-osteogenic system, characterized in that, include: Mesoporous silica nanoparticles were added to a dexamethasone ethanol solution, stirred for more than 24 hours in the dark, centrifuged, washed, and freeze-dried to obtain dexamethasone-loaded nanoparticles. The dexamethasone-loaded nanoparticles were dispersed in a buffer solution, and then dopamine hydrochloride was added. The mixture was stirred in the dark for more than 12 hours, centrifuged, washed, and freeze-dried to obtain polydopamine-coated dexamethasone-loaded nanoparticles. The polydopamine-coated dexamethasone nanoparticles were added to a BMP-2 solution, stirred for more than 4 hours, centrifuged, and freeze-dried to obtain polydopamine-coated dexamethasone and BMP-2 mesoporous silica nanoparticles. The mass ratio of the mesoporous silica nanoparticles to dexamethasone is 100:6-10; The mass ratio of the polydopamine-coated dexamethasone nanoparticles to BMP-2 is 100:0.012-0.

018.

2. The method for preparing the pH-responsive polydopamine-coated dexamethasone and BMP-2 mesoporous silica nano-osteogenic system as described in claim 1, characterized in that, The mass ratio of the mesoporous silica nanoparticles to dexamethasone is 100:

8.

3. The method for preparing the pH-responsive polydopamine-coated dexamethasone and BMP-2 mesoporous silica nano-osteogenic system as described in claim 1, characterized in that, The concentration of the dexamethasone ethanol solution is 2-6 mg / mL.

4. The method for preparing the pH-responsive polydopamine-coated dexamethasone and BMP-2 mesoporous silica nano-osteogenic system as described in claim 3, characterized in that, The concentration of the dexamethasone ethanol solution was 4 mg / mL.

5. The method for preparing the pH-responsive polydopamine-coated dexamethasone and BMP-2 mesoporous silica nano-osteogenic system as described in claim 1, characterized in that, The method for preparing the mesoporous silicon nanoparticles includes: Step 1: Mix hexadecyltrimethylammonium bromide and sodium hydroxide with water until homogeneous, heat and stir continuously until completely dissolved; then add tetraethyl orthosilicate to react, centrifuge and wash to obtain the first product; Step 2: Add the first product to an ethanol solution containing HCl, reflux the reaction, centrifuge, wash, and collect the second product; Step 3: Repeat step 2 multiple times to remove the template agent hexadecyltrimethylammonium bromide. Then, centrifuge, wash, and dry to obtain mesoporous silica nanoparticles.

6. The method for preparing the pH-responsive polydopamine-coated dexamethasone and BMP-2 mesoporous silica nano-osteogenic system as described in claim 1, characterized in that, The mass concentration of the dexamethasone-loaded nanoparticles in the buffer solution is 1-4 mg / mL.

7. The method for preparing the pH-responsive polydopamine-coated dexamethasone and BMP-2 mesoporous silica nano-osteogenic system as described in claim 6, characterized in that, The mass concentration of the dexamethasone-loaded nanoparticles in the buffer solution was 2 mg / mL.

8. The method for preparing the pH-responsive polydopamine-coated dexamethasone and BMP-2 mesoporous silica nano-osteogenic system as described in claim 1, characterized in that, The mass ratio of the polydopamine-coated dexamethasone nanoparticles to BMP-2 is 100:0.

016.

9. The method for preparing the pH-responsive polydopamine-coated dexamethasone and BMP-2 mesoporous silica nano-osteogenic system as described in claim 1, characterized in that, The concentration of the BMP-2 solution is 2-6 μg / mL.

10. The method for preparing the pH-responsive polydopamine-coated dexamethasone and BMP-2 mesoporous silica nano-osteogenic system as described in claim 9, characterized in that, The concentration of the BMP-2 solution was 4 μg / mL.

11. The pH-responsive polydopamine-coated dexamethasone and BMP-2 mesoporous silica nano-osteogenic system prepared by the method of any one of claims 1-10.

Images