Slow-release docetaxel nano-composite as well as preparation method and application thereof

Abstract

The invention relates to a sustained-release docetaxel nano-composite and a preparation method and application thereof, and the preparation method comprises the following steps: S1, uniformly mixing docetaxel with a mesoporous silica nano-particle dispersion liquid to obtain a docetaxel-loaded mesoporous silica nano-composite, which is recorded as DTX-coated MSN; and S2, coating the surface of the DTX (at) MSN with branched polyethyleneimine through surface self-assembly, so as to obtain the slow-release docetaxel nano-composite, which is recorded as DTX (at) MSN-PEI. Compared with the prior art, the docetaxel nano-composite provided by the invention can improve the action efficiency of inducing cartilage differentiation of stem cells by a drug through slow release of the drug in cells, and has important significance and potential application value for promoting clinical transformation of MSCs for treating OA cartilage injury.

Description

Technical Field

[0001] The present invention relates to the technical field of nanobiomaterials, and particularly relates to a sustained-release docetaxel nanocomplex, a preparation method thereof, and an application thereof. Background Art

[0002] Osteoarthritis (OA) is a chronic joint disease with the structural destruction of articular surface hyaline cartilage as the main pathological feature. Due to the lack of blood vessels and nerve distribution in articular cartilage, its self-repair ability is poor. Clinically, oral or intra-articular injection of anti-inflammatory drugs and cytokines are mostly adopted to delay the progression of articular cartilage degeneration, but the prognosis effect is not good. At present, there is no effective means to cure cartilage damage of osteoarthritis. In recent years, the emerging stem cell therapy has brought new hope for repairing cartilage damage of osteoarthritis: on the one hand, by recruiting mesenchymal stem cells (MSCs) in joint synovial fluid or bone marrow blood through cytokines to migrate to the damaged site, the regenerative repair of cartilage defects can be achieved by using the chondrogenic ability of MSCs; on the other hand, in order to make up for the limitation of the insufficient number of endogenous MSCs, MSCs preparations for in vivo transplantation are developed through an in vitro engineering platform, which also has great clinical application potential. However, the ability of MSCs recruited in vivo or transplanted in vitro to differentiate into hyaline chondrocytes and secrete matrix in the OA microenvironment is significantly weakened, which greatly limits the basic research and clinical transformation of MSCs in the treatment of OA.

[0003] Docetaxel (DTX) is a class of cell cycle-specific small molecule drugs. Its stabilizing effect on the synthesis of intracellular microtubule structure has been widely recognized. It has not only been approved by the FDA for clinical chemotherapy of tumors, but also shown unique application potential in tissue regeneration because it can promote axon growth of nerve cells and enhance matrix secretion of hyaline chondrocytes. However, due to the short in vivo half-life and easy inactivation of small molecule drugs, the improvement effect of injecting or orally delivering docetaxel into the joint cavity on enhancing the ability of MSCs to repair cartilage damage of osteoarthritis is extremely limited. Summary of the Invention

[0004] The purpose of the present invention is to provide a sustained-release docetaxel nanocomplex, a preparation method thereof, and an application thereof to overcome at least one of the defects existing in the above-mentioned prior art. By the site-specific modification of MSCs with docetaxel, the ability of MSCs to differentiate into hyaline chondrocytes can be more effectively enhanced, further promoting the clinical transformation of MSCs in the treatment of OA diseases.

[0005] The purpose of the present invention can be realized by the following technical solutions:

[0006] One of the objectives of the present invention is a method for preparing a sustained-release docetaxel nanocomplex, comprising the following steps:

[0007] S1. Mix docetaxel evenly with a dispersion of mesoporous silica nanoparticles (MSN) to obtain a mesoporous silica nanocomplex loaded with docetaxel, denoted as DTX@MSN;

[0008] S2. Coating branched polyethyleneimine (bPEI) on the surface of DTX@MSN through surface self-assembly to obtain a sustained-release docetaxel nanocomplex, denoted as DTX@MSN-PEI.

[0009] Furthermore, the preparation method of the mesoporous silica nanoparticles is as follows: Prepare an aqueous solution of cetyltrimethylammonium chloride (CTAC) containing triethanolamine, and dropwise add a silane coupling agent while stirring under heating and reflux conditions. After the reaction, centrifuge to collect the precipitate, and remove cetyltrimethylammonium chloride in the precipitate to obtain mesoporous silica nanoparticles.

[0010] Furthermore, the concentration of the cetyltrimethylammonium chloride aqueous solution is 10% (w / v) - 15% (w / v), and the volume fraction of the contained triethanolamine is 0.1% (v / v) - 0.5% (v / v).

[0011] Furthermore, the reflux temperature is 80°C - 100°C.

[0012] In the present invention, the silane coupling agent satisfies at least one of the following conditions:

[0013] ① The silane coupling agent is tetraethyl orthosilicate;

[0014] ② The mass ratio of the silane coupling agent to the cetyltrimethylammonium chloride is 0.5:1 - 1:1;

[0015] ③ The dropping rate of the silane coupling agent is 0.1 mL / min - 0.15 mL / min.

[0016] Furthermore, cetyltrimethylammonium chloride in the precipitate is removed by repeatedly washing with a sodium chloride-methanol solution for multiple times, and the concentration of the sodium chloride-methanol extraction solution is 1% (w / v) - 3% (w / v).

[0017] Furthermore, the mass ratio of the docetaxel to the mesoporous silica nanoparticles is (0 - 0.75) μg:1 mg.

[0018] Furthermore, in S2, the concentration of DTX@MSN is 1 mg / mL - 2 mg / mL, and the concentration of the branched polyethyleneimine is 0.5 mg / mL - 1.0 mg / mL.

[0019] The second object of the present invention is a sustained-release docetaxel nanocomplex prepared by the preparation method as described above.

[0020] The third object of the present invention is the application of a sustained-release docetaxel nanocomplex as described above in inducing the differentiation of mesenchymal stem cells into chondrocytes;

[0021] Among them, the mesenchymal stem cells include any one or more of bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, and synovial joint stem cells.

[0022] Preferably, the monolayer starting cell density required for inducing chondrogenic differentiation of MSCs is 0.5×10 4 cell / cm 2 ~2×10 4 cell / cm 2 ; the mass-volume concentration of the DTX@MSN-PEI nanocomplex required is 100 μg / mL to 200 μg / mL; the incubation time of the DTX@MSN-PEI nanocomplex is 12 to 48 hours.

[0023] Compared with the prior art, the docetaxel nanocomplex provided by the present invention can realize the functional modification of small molecule drugs on MSCs through the slow release of drugs in cells, improve the efficiency of drug-induced chondrogenic differentiation of stem cells, and has important significance and potential application value for promoting the clinical transformation of MSCs in the treatment of OA cartilage damage. Description of the Drawings

[0024] Figure 1 It is the result diagram of the basic physicochemical properties of the docetaxel nanocomplex: (A) Transmission electron microscopy result diagram; (B) Hydrodynamic particle size and ζ potential result diagram.

[0025] Figure 2 It is the result diagram of the cumulative release rate of the docetaxel nanocomplex at different time points.

[0026] Figure 3 It is the result diagram of the cell uptake of the fluorescently labeled nanocomplex.

[0027] Figure 4 It is the result diagram of the cell uptake mechanism of the fluorescently labeled nanocomplex.

[0028] Figure 5 It is the result diagram of the effect of the docetaxel nanocomplex on inducing chondrogenic differentiation of BMSCs detected by real-time fluorescence quantitative PCR; among them, * p < 0.05, ** p < 0.01, *** p < 0.001. Detailed Embodiments

[0029] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation procedures are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0030] The present invention provides a method for preparing a sustained-release docetaxel nanocomposite, which includes the following steps:

[0031] S1. Mix docetaxel evenly with a mesoporous silica nanoparticle (MSN) dispersion to obtain a mesoporous silica nanocomposite loaded with docetaxel, denoted as DTX@MSN;

[0032] S2. Coating branched polyethyleneimine (bPEI) on the surface of DTX@MSN through surface self-assembly to obtain a sustained-release docetaxel nanocomposite, denoted as DTX@MSN-PEI.

[0033] In some embodiments of the present invention, the method for preparing the mesoporous silica nanoparticles is as follows: Prepare an aqueous solution of cetyltrimethylammonium chloride (CTAC) containing triethanolamine, and dropwise add a silane coupling agent while stirring under heating and reflux conditions. After the reaction, centrifuge to collect the precipitate, and remove the cetyltrimethylammonium chloride in the precipitate to obtain mesoporous silica nanoparticles.

[0034] In some embodiments of the present invention, the concentration of the cetyltrimethylammonium chloride aqueous solution is 10% (w / v) - 15% (w / v), and the volume fraction of the contained triethanolamine is 0.1% (v / v) - 0.5% (v / v).

[0035] In some embodiments of the present invention, the reflux temperature is 80°C - 100°C.

[0036] In some embodiments of the present invention, the silane coupling agent satisfies at least one of the following conditions:

[0037] In the present invention, the silane coupling agent is tetraethyl orthosilicate;

[0038] In the present invention, the mass ratio of the silane coupling agent to the cetyltrimethylammonium chloride is 0.5:1 - 1:1.

[0039] In the present invention, the dropping rate of the silane coupling agent is 0.1 mL / min - 0.15 mL / min.

[0040] In some embodiments of the present invention, the cetyltrimethylammonium chloride in the precipitate is removed by repeatedly washing with a sodium chloride - methanol solution, and the concentration of the sodium chloride - methanol extract is 1% (w / v) to 3% (w / v).

[0041] In some embodiments of the present invention, the mass ratio of docetaxel to the mesoporous silica nanoparticles is (0 - 0.75) μg:1 mg.

[0042] In some embodiments of the present invention, in S2, the concentration of DTX@MSN is 1 mg / mL to 2 mg / mL, and the concentration of branched polyethyleneimine is 0.5 mg / mL to 1.0 mg / mL.

[0043] The present invention also provides a sustained - release docetaxel nanocomposite prepared by the preparation method described above.

[0044] The present invention also provides an application of the sustained - release docetaxel nanocomposite described above in inducing the differentiation of mesenchymal stem cells into hyaline chondrocytes;

[0045] Among them, the mesenchymal stem cells include any one or more of bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, and synovial joint stem cells; the monolayer starting cell density required for inducing chondrogenic differentiation of MSCs is 0.5×10 4 cell / cm 2 ~2×10 4 cell / cm 2 ; the mass - volume concentration of the DTX@MSN - PEI nanocomposite required is 100 μg / mL to 200 μg / mL; the incubation time of the DTX@MSN - PEI nanocomposite is 12 to 48 hours.

[0046] Example 1

[0047] This example provides a preparation method of a docetaxel nanocomposite, which specifically includes the following steps:

[0048] S1. Prepare 20 mL of a 10% (w / v) CTAC aqueous solution containing 0.36% (v / v) triethanolamine, continuously reflux at 95 °C for 1 hour, dropwise add the silane coupling agent TEOS at a constant rate of 0.1 mL / min, after continuously reacting for 1 hour, centrifuge to collect the precipitate, repeatedly wash with a 1% (w / v) sodium chloride - methanol solution several times, and disperse the obtained product, namely mesoporous silica nanoparticles (MSN), in an ethanol medium for standby, thus obtaining an MSN dispersion.

[0049] S2. Add 1 μL of 1 mmol / L docetaxel to 1 mL of 4 mg / mL MSN dispersion, stir overnight at room temperature, and obtain docetaxel-mesoporous silica nanocomposite by centrifugation and washing, denoted as DTX(0.8)@MSN. Meanwhile, collect the centrifuged supernatant to detect the drug encapsulation efficiency of docetaxel.

[0050] For comparison, add 3.75 μL of 1 mmol / L docetaxel to 1 mL of 4 mg / mL MSN dispersion, stir overnight at room temperature, and obtain docetaxel-mesoporous silica nanocomposite by centrifugation and washing, denoted as DTX(3)@MSN.

[0051] S3. Mix 0.5 mL of 8 mg / mL DTX@MSN dispersion with 2 mL of 1 mg / mL bPEI solution under ultrasonic oscillation conditions, stir well, and collect the product by centrifugation. The products are denoted as DTX(0.8)@MSN-PEI and DTX(3)@MSN-PEI respectively.

[0052] For comparison, mix 0.5 mL of 8 mg / mL MSN dispersion with 2 mL of 1 mg / mL bPEI solution under ultrasonic oscillation conditions, stir well, and collect the product by centrifugation. The product is denoted as DTX(0)@MSN-PEI.

[0053] Please refer to Figure 1 , the hydrodynamic diameters of DTX(0)@MSN-PEI, DTX(0.8)@MSN-PEI, and DTX(3)@MSN-PEI nanoparticles are (181.54 ± 2.89) nm, (232.27 ± 6.55) nm, and (188.7 ± 4.92) nm respectively, and the ζ potentials are (-33.17 ± 1.66) mV, (18.29 ± 0.21) mV, and (23.03 ± 2.32) mV in sequence, indicating that the docetaxel nanocomposites obtained by the preparation method provided in this example have good dispersibility in physiological solutions.

[0054] Example 2

[0055] This example provides a method for detecting the drug encapsulation efficiency and cumulative release rate of docetaxel nanocomposite.

[0056] In this example, the drug encapsulation efficiency of the nanocomplex was calculated by detecting the drug concentration in the supernatant of the encapsulation, and the cumulative drug release rate of the nanocomplex was calculated by detecting the free drug concentration of the docetaxel nanocomplex in physiological saline at different time points. The detection of the drug concentration was carried out by means of an ultra-high performance liquid separation chromatography-triple quadrupole mass spectrometer (manufacturer: WATERS; model: AB5500). The specific test conditions were as follows: C18 chromatographic column, gradient elution with acetonitrile-water polar solvents, and column temperature of 40 °C.

[0057] The docetaxel nanocomplex obtained by the preparation method provided in Example 1 had a relatively high drug encapsulation efficiency ((75.0 ± 1.72)%), and the cumulative release rates at 12 hours, 24 hours, and 48 hours were (24.8 ± 0.027)%, (25.2 ± 0.108)%, and (25.3 ± 0.054)%, respectively, indicating that the nanocomplex presented a stable drug release behavior, as shown in Figure 2 .

[0058] Example 3

[0059] In this example, the cellular uptake behavior and endocytic pathway of the docetaxel nanocomplex were evaluated by fluorescence colocalization.

[0060] In this example, bone marrow mesenchymal stem cells (BMSCs) were used for experimental verification. Mesoporous silica nanocomposites (FITC@MSN-PEI) loaded with fluorescein isothiocyanate (FITC) were synthesized by the preparation method provided in Example 1, that is, the docetaxel in Example 1 was replaced with FITC. BMSCs were seeded on the surface of cell slides at a cell density of 1 × 10 4 cell / cm 2 . After the cells adhered, the growth medium containing 150 μg / mL FITC@MSN-PEI was replaced and incubated for 24 hours and 48 hours, respectively. After fixation with 4% paraformaldehyde solution, DAPI staining solution was used for counterstaining for 5 minutes. After mounting with glycerol, microscopic examination was carried out, as shown in Figure 3 .

[0061] It was found that Figure 3 with the extension of the incubation time, the intracellular fluorescence intensity of BMSCs gradually increased, indicating that the uptake of the nanocomplex by BMSCs presented a time-dependent kinetic process.

[0062] In this example, BMSCs were pretreated with 50 μmol / L amiloride (macropinocytosis inhibitor), 14 μmol / L chlorpromazine (clathrin inhibitor), and 5 mmol / L methyl-β-cyclodextrin (caveolin inhibitor), respectively, and then incubated with a growth medium containing 150 μg / mL FITC@MSN-PEI for 48 hours. After fixation with 4% paraformaldehyde solution, DAPI staining solution was used for counterstaining for 5 minutes, and after mounting with glycerol, microscopic examination was carried out( Figure 4 ).

[0063] As Figure 4 can be seen, the intracellular fluorescence intensity of BMSCs in the chlorpromazine treatment group was significantly weakened, indicating that clathrin-mediated endocytosis may be the main pathway for BMSCs to uptake the docetaxel nanocomplex. Lysosomes, endosomes and other organelles are usually considered to be the main transport pathways for clathrin-mediated endocytosis. Therefore, the mesoporous silica nanocomplex provided in this example can achieve effective transport of docetaxel from extracellular to cytoplasm.

[0064] Example 4

[0065] In this example, RT-qPCR was used to quantitatively analyze the effect of docetaxel nanocomplex on the chondrogenic differentiation of BMSCs in Example 1.

[0066] In this example, a chondrogenic induction medium containing 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 1 mmol / L sodium pyruvate, insulin-transferrin-selenium additive (10 μg / mL recombinant human insulin, 5.5 μg / mL transferrin, 6.7 ng / mL sodium selenite), 40 ng / mL dexamethasone, 50 μg / mL L-ascorbic acid, 46 μg / mL L-proline, and 10 ng / mL TGF-β1 was prepared. After pretreatment of BMSCs with the DTX@MSN-PEI nanocomplex, the chondrogenic induction medium was used to continue incubation for 7 days.

[0067] The analysis method in this example was as follows: RNA was extracted using a total RNA extraction kit (stored at -80 °C for later use), and mRNA was reverse transcribed into cDNA according to the reaction system, operation steps, and reverse transcription conditions shown in Table 1.

[0068] Table 1 Settings of reagents, reaction system, and reverse transcription program for RNA reverse transcription

[0069]

[0070] Furthermore, after diluting the obtained reverse transcription product by 10 times, quantitative PCR reaction was carried out according to the reaction system and amplification program shown in Table 2, and the melting curve was recorded and analyzed.

[0071] Table 2 Quantitative PCR reaction system and amplification program

[0072]

[0073] Table 3 Primer sequences

[0074]

[0075] By Figure 5 It can be found that, compared with the control group, the pretreatment process of DTX(0.8)@MSN-PEI nanocomposites significantly increased the expression of Col II, SOX9, and ACAN markers in BMSCs, indicating that the docetaxel nanocomposites provided by the present invention can improve the chondrogenic differentiation efficiency of BMSCs.

[0076] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention in any other form. Any person skilled in the art may use the technical content disclosed above to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a sustained-release docetaxel nanocomposite, characterized in that: The steps include: S1, mixing docetaxel and a dispersion of mesoporous silica nanoparticles evenly to obtain a mesoporous silica nanocomposite loaded with docetaxel, denoted as DTX@MSN; S2. Branched polyethyleneimine was coated on the surface of DTX@MSN by surface self-assembly to obtain a sustained-release docetaxel nanocomposite, which was recorded as DTX@MSN-PEI.

2. The method for preparing a sustained-release docetaxel nanocomposite according to claim 1, characterized in that: The preparation method of the mesoporous silica nanoparticles comprises: preparing a hexadecyltrimethylammonium chloride aqueous solution containing triethanolamine, adding a silane coupling agent dropwise under heating reflux conditions while stirring, collecting a precipitate by centrifugation after the reaction, removing the hexadecyltrimethylammonium chloride in the precipitate, and obtaining the mesoporous silica nanoparticles.

3. The method for preparing a sustained-release docetaxel nanocomposite according to claim 2, characterized in that: The concentration of the hexadecyltrimethylammonium chloride aqueous solution is 10% (w / v) to 15% (w / v), and the volume fraction of triethanolamine contained therein is 0.1% (v / v) to 0.5% (v / v).

4. The method for preparing a sustained-release docetaxel nanocomposite according to claim 2, characterized in that: The reflux temperature is 80°C to 100°C.

5. The method for preparing a sustained-release docetaxel nanocomposite according to claim 2, characterized in that: The silane coupling agent satisfies at least one of the following conditions: ① The silane coupling agent is tetraethyl orthosilicate; ② The mass ratio of the silane coupling agent to the hexadecyltrimethylammonium chloride is 0.5:1 to 1:1; ③ The dropping rate of the silane coupling agent is 0.1 mL / min to 0.15 mL / min.

6. The method for preparing a sustained-release docetaxel nanocomposite according to claim 2, characterized in that: The hexadecyltrimethylammonium chloride in the precipitate is removed by repeated washing with a sodium chloride-methanol solution, wherein the concentration of the sodium chloride-methanol extract is 1% (w / v) to 3% (w / v).

7. The method for preparing a sustained-release docetaxel nanocomposite according to claim 1, characterized in that: The mass ratio of the docetaxel to the mesoporous silica nanoparticles is (0-0.75) μg:1 mg.

8. The method for preparing a sustained-release docetaxel nanocomposite according to claim 1, characterized in that: In S2, the concentration of the DTX@MSN is 1 mg / mL to 2 mg / mL, and the concentration of the branched polyethyleneimine is 0.5 mg / mL to 1.0 mg / mL.

9. A sustained-release docetaxel nanocomposite, characterized in that: The invention discloses a novel nanostructured carbon foam. The nanostructured carbon foam is prepared by the preparation method according to any one of claims 1 to 8.

10. A use of the sustained-release docetaxel nanocomposite as claimed in claim 9, characterized in that: Applied in inducing mesenchymal stem cells to differentiate into hyaline chondrocytes; The mesenchymal stem cells include any one or more of bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, and joint synovial stem cells.

Images