Mesoporous silica nanoparticles, their preparation methods, pharmaceutical compositions and applications

Abstract

This invention discloses a mesoporous silica nanoparticle, its preparation method, pharmaceutical composition, and applications. The nanoparticle is epoxy-modified, with its mesoporous channels loaded with polypeptide precursor molecules grafted onto chemotherapeutic drug molecules. Its outer layer is coated with an acid-sensitive polymer layer, enabling it to trigger drug release in the slightly acidic environment of the tumor. Furthermore, under the catalysis of highly expressed active enzymes within the nanoparticle, in-situ gelation occurs at the lesion site, thereby prolonging the retention time of the nanomedicine, improving the bioavailability of chemotherapeutic drugs, and enhancing the efficacy of chemotherapy.

Description

Technical Field

[0001] This invention relates to mesoporous silica nanoparticles, their preparation methods, pharmaceutical compositions, and applications, and particularly to mesoporous silica nanoparticles with enzyme-responsive in-situ gelation properties, their preparation methods, pharmaceutical compositions, and applications. Background Technology

[0002] Chemotherapy is a common treatment for cancer, but its efficacy is limited by various factors, such as the effective concentration and duration of drug delivery at the tumor site, nonspecific toxicity, and drug resistance. While nanomedicine delivery systems can improve drug targeting and bioavailability, they still suffer from problems such as uncontrollable drug release and short residence time.

[0003] Mesoporous silica nanoparticles (MSNs), due to their large specific surface area, good biocompatibility, and tunable pore structure, can effectively load and protect chemotherapeutic drug molecules, enabling controlled drug release at cancer lesions. However, traditional MSN drug delivery systems still suffer from problems such as difficulty in controlling drug release rates and insufficient drug retention time in target tissues. Therefore, there is currently no effective drug delivery system that solves these problems. Summary of the Invention

[0004] Purpose of the invention: The first purpose of this invention is to provide mesoporous silica nanoparticles with specific responsiveness and in-situ gelling properties; the second purpose is to provide a method for preparing the nanoparticles; the third purpose is to provide a pharmaceutical composition and molecular probe composed of the nanoparticles; and the fourth purpose is to provide a pharmaceutical application of the nanoparticles, the pharmaceutical composition, and the molecular probe.

[0005] Technical solution: The mesoporous silica nanoparticles of the present invention, wherein the nanoparticles are epoxy-modified, and their mesoporous channels are loaded with polypeptide precursor molecules grafted with chemotherapy drug molecules, and their outer layer is coated with an acid-sensitive polymer layer.

[0006] The tumor microenvironment typically exhibits low pH and high levels of enzyme biomarkers, characteristics that can serve as signals to trigger drug release. Therefore, designing a nanocarrier capable of responding to specific conditions within the tumor microenvironment could help improve the efficacy of chemotherapy and reduce side effects.

[0007] On the one hand, by improving the performance of MSNs through surface modification and functionalization, and introducing enzyme response and acid sensitivity properties, the drug release mechanism can be further optimized.

[0008] On the other hand, peptide hydrogel drug delivery systems with enzymatic self-assembly properties can significantly improve the retention time and bioavailability of chemotherapeutic drugs at the tumor site. For example, peptide molecules with phenylalanine-phenylalanine-phosphorylated tyrosine (FFYp) structural units can undergo dephosphorylation under the action of phosphatases highly expressed in tumor tissue, triggering the self-assembly of peptide molecules to form a three-dimensional network hydrogel structure, thereby accumulating at the lesion site. Furthermore, the multifunctionality of peptide molecules allows for the grafting of various chemotherapeutic drugs, thus achieving long-term retention and slow release of these drugs. Therefore, integrating these functions into MSNs can significantly improve their application efficacy in tumor treatment.

[0009] This invention designs an enzyme-responsive, in-situ gelling mesoporous silica nanoparticle. By integrating enzyme responsiveness and acid sensitivity, it achieves controlled drug release and in-situ gelation, thereby prolonging the drug's residence time in the lesion area, improving the bioavailability of chemotherapy drugs, and ultimately enhancing the chemotherapy effect. Specifically, peptide precursor molecules are loaded into the mesoporous channels of the mesoporous silica nanoparticles to form a hydrogel. The gelling ability of the peptide precursor molecules can be regulated by the peptide sequence, and chemotherapy drug molecules are grafted onto the side chains of the peptides. Simultaneously, an acid-sensitive polymer layer is modified on the outer layer of the mesoporous silica nanoparticles to achieve controlled drug release.

[0010] Preferably, the polypeptide precursor molecule is selected from one or more of Nap-Phe-Phe-Tyr(H2PO3)-Lys-OH, Nap-Phe-Tyr(H2PO3)-Lys-OH, Phe-Phe-Tyr(H2PO3)-Lys-OH, Val-Cit-Phe-Phe-Lys-OH, Ala-Phe-Phe-Lys-OH, Phe-Tyr(H2PO3)-Lys-OH, and Lys-Tyr(H2PO3)-Phe-Phe-OH, wherein the Lys amino acid side is branched with a chemotherapeutic drug molecule. The polypeptide precursor molecule can be cleaved by highly expressed active enzymes in the tumor microenvironment, exposing gel-forming factors, which then undergo in-situ self-assembly under the influence of intermolecular forces and hydrogen bonds to form a three-dimensional network hydrogel structure. This hydrogel network can immobilize mesoporous silica nanoparticles at the tumor lesion site, thereby prolonging their retention time and further enhancing their in-situ gelation effect. The active enzymes include one or more of cathepsin B, aminopeptidase N, alkaline phosphatase, and acid phosphatase.

[0011] Preferably, the chemotherapeutic drug molecule is selected from one or more of paclitaxel, camptothecin, and doxorubicin. After release, the polypeptide precursor molecule further activates the loaded chemotherapeutic drug within the cell, exerting its therapeutic effect and killing tumor cells.

[0012] Preferably, the acid-sensitive polymer is selected from one or more of poly(N-isopropylacrylamide), polyethylene glycol-poly(α-hydroxy acid) block copolymer, chitosan, and polymethacrylate. The acid-sensitive polymer is rapidly protonated in the microacidic environment of a tumor, thereby releasing the loaded polypeptide precursor molecules.

[0013] Preferably, the nanoparticles have a particle size of 100-120 nm and a PDI < 0.2.

[0014] The method for preparing mesoporous silica nanoparticles according to the present invention includes the following steps:

[0015] (1) Preparation of epoxy-modified mesoporous silica nanoparticles;

[0016] (2) The nanoparticles prepared in step (1) are coated with acid-sensitive polymer;

[0017] (3) The nanoparticles prepared in step (2) are loaded with chemotherapeutic drug molecules and polypeptide precursor molecules in the mesoporous channels.

[0018] The specific methods for each step are as follows:

[0019] (1) Preparation of epoxy-modified mesoporous silica nanoparticles

[0020] a. Mesoporous silica nanoparticles are prepared using an oil-water two-phase method. The oil phase consists of one or more of chloroform, octadecene, n-hexane, decahydronaphthalene, and cyclohexane; the catalyst consists of one or more of concentrated ammonia, triethanolamine, sodium hydroxide, potassium hydroxide, and triethylamine; and the surfactant consists of one or more of hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium chloride, and dodecyltrimethylammonium bromide. The catalyst and surfactant are dissolved in water, and the oil phase is dropped onto the surface of the aqueous solution. An appropriate amount of one or more of tetraethyl orthosilicate, methyl orthosilicate, or propyl orthosilicate is mixed into the oil phase as a silicon source. The amounts of the catalyst, surfactant, and silicon source are all 0.5% to 10% of the total mass. The reaction is carried out at 40–90°C for 6–24 hours, followed by centrifugation or filtration.

[0021] b. Disperse the sample obtained in the previous step in hydrochloric acid-ethanol solution (2% v / v), reflux at 70°C for 12 h, then centrifuge or filter, repeat 3 times.

[0022] c. Dry the sample obtained in the previous step, disperse it in toluene, add γ-glycidoxypropyltrimethoxysilane, and carry out the reaction at 60-110℃ for 6-24 hours. After centrifugation or filtration, epoxy-modified mesoporous silica nanoparticles can be obtained.

[0023] The above methods can be used to prepare materials with a size of 20–200 nm, a pore size of 3–15 nm, and a specific surface area of ​​200–900 m². 2 / g of mesoporous silica nanoparticles.

[0024] (2) Coating with acid-sensitive polymer layers

[0025] The acid-sensitive polymer is selected from one or more of poly(N-isopropylacrylamide), polyethylene glycol-poly(α-hydroxy acid) block copolymer, chitosan, and polymethacrylate. The epoxy-modified mesoporous silica nanoparticles obtained in the previous step are dispersed in ethanol, and the acid-sensitive polymer is added; the mass ratio of the acid-sensitive polymer to the mesoporous silica nanoparticles is 1:10 to 1:1. The reaction is carried out at 25–50 °C for 2–6 h, followed by centrifugation or filtration, and repeated three times.

[0026] (3) Loading of peptide precursor molecules

[0027] The mesoporous silica nanoparticles coated with an acid-sensitive polymer layer from the previous step were dispersed in dimethyl sulfoxide (DMSO). A peptide precursor molecule was added, with a mass ratio of peptide precursor to mesoporous silica nanoparticles of 1:10 to 1:1. The solution was placed in a water bath at 25–40°C for 2–6 hours. Phosphate-buffered saline (PBS, pH 7.4) was then added, and stirring continued for 10–30 minutes. The mixture was then centrifuged or filtered, and washed three times with PBS to obtain enzyme-responsive in-situ gel-forming mesoporous silica nanoparticles.

[0028] The specific preparation method of the polypeptide precursor molecule is as follows:

[0029] A certain amount of amino acid monomers are weighed and sequentially linked according to the set polypeptide sequence. The polypeptide chain is then constructed stepwise on the synthetic resin using a solid-phase peptide synthesis method. After synthesis, the polypeptide is released from the synthetic resin by a cleavage reaction and purified by high-performance liquid chromatography (HPLC) to obtain the desired polypeptide precursor molecule.

[0030] The specific method for branching and grafting the polypeptide precursor onto the chemotherapeutic drug molecule is as follows:

[0031] The purified peptide precursor molecules were dissolved in a suitable solvent and grafted onto the chemotherapeutic drug molecules under stirring conditions to ensure successful grafting of the drug molecules onto the peptide side chains. The resulting peptide precursor molecules were then purified again by high-performance liquid chromatography (HPLC) to ensure purity. The purified peptide precursor molecules were then dried under freeze-drying conditions and stored for later use.

[0032] The pharmaceutical composition of the present invention comprises the mesoporous silica nanoparticles of the present invention and a pharmaceutically acceptable carrier.

[0033] The molecular probe described in this invention is obtained by loading fluorescent dyes onto mesoporous silica nanoparticles as described in this invention.

[0034] The mesoporous silica nanoparticles and their pharmaceutical compositions or molecular probes described in this invention are used in the preparation of drugs for treating tumors.

[0035] Preferably, the mesoporous silica nanoparticles and their drug compositions or molecular probes undergo an in-situ gelation reaction catalyzed by one or more enzymes selected from cathepsin B, aminopeptidase N, alkaline phosphatase, and acid phosphatase.

[0036] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0037] The enzyme-responsive in-situ gelling mesoporous silica nanoparticles designed in this invention exhibit strong targeting capabilities. Through their enzyme-responsive and acid-sensitive properties, they ensure the precise release of peptide precursor molecules grafted with chemotherapeutic drugs at the tumor site. Simultaneously, the in-situ gelling strategy significantly prolongs the residence time of the carrier and drug in the lesion area, and also increases the effective concentration of chemotherapeutic drugs at the tumor site, enhancing therapeutic efficacy. This effectively solves the problems of short drug residence time, high toxicity, and low bioavailability in existing chemotherapy methods, thereby achieving more precise and safer enhanced chemotherapy. Attached Figure Description

[0038] Figure 1 A schematic diagram of the structure of mesoporous silica nanoparticles;

[0039] Figure 2 Transmission electron microscope image of Nap-FYp-CPT@MSN-CHI;

[0040] Figure 3 This is a synthetic route diagram for polypeptide precursor molecules;

[0041] Figure 4 The structural characterization map of the polypeptide precursor molecule;

[0042] Figure 5 Photographs showing the in vitro in-situ gelation properties of mesoporous silica nanoparticles loaded with peptide precursor molecules.

[0043] Figure 6 The killing effect of enzyme-responsive in-situ gel-forming mesoporous silica nanoparticles on tumor cells is illustrated.

[0044] Figure 7 The in vivo long retention of enzyme-responsive in situ gel-forming mesoporous silica nanoparticles and their enhanced chemotherapy effect are illustrated.

[0045] Figure 8 This is a schematic diagram illustrating the drug release mechanism of mesoporous silica nanoparticles. Detailed Implementation

[0046] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0047] Example 1: Synthesis and purification of mesoporous silica

[0048] A 100 mL round-bottom flask was used as the reaction vessel. First, 6 g of hexadecyltrimethylammonium bromide and 0.18 g of triethanolamine were weighed and placed in the flask. Then, 54 mL of deionized water was added, and the mixture was sonicated for 30 min. The mixture was then stirred in a 60 °C water bath for 1 h. Subsequently, a tetraethoxysilane-cyclohexane solution (20% v / v) was slowly added along the flask wall, and the reaction continued in a 60 °C water bath for 12 h. After the reaction was complete, the upper cyclohexane layer was removed, and the lower milky white turbid liquid was collected and centrifuged and washed three times. The sample obtained in the previous step was dispersed in a hydrochloric acid-ethanol solution (2% v / v), refluxed at 70 °C for 12 h, and centrifuged. This process was repeated three times.

[0049] Example 2: Synthesis and purification of epoxy-modified mesoporous silica

[0050] The sample obtained in Example 1 was dried, dispersed in 20 mL of toluene, and 100 μL of γ-glycidoxypropyltrimethoxysilane was added. After reacting at 110 °C for 10 h, the sample was centrifuged and washed three times.

[0051] Example 3: Synthesis and purification of chitosan-modified mesoporous silica

[0052] The epoxy-modified mesoporous silica obtained in Example 2 was dispersed in 2 mL of ethanol solution, 5 mL of chitosan solution and 50 μL of triethylamine solution were added, and the mixture was reacted in a water bath at 37 °C for 24 h. After the reaction was completed, the sample was centrifuged, washed three times and dried.

[0053] Example 4: Synthesis and purification of the peptide prodrug Nap-FYp-CPT

[0054] (1) Synthesis of carboxylated camptothecin (CPT)

[0055] First, 25 mL of pyridine was measured as the reaction solution. Then, 0.89 g of CPT, 0.77 g of succinic anhydride, and 31 mg of 4-dimethylaminopyridine were weighed and added to the pyridine solution. The mixture was stirred for 4 h at 80 °C under nitrogen protection. After the reaction was completed, the mixture was washed three times each with 1% hydrochloric acid aqueous solution and pure aqueous solution and dried to obtain purified carboxylated CPT.

[0056] (2) Synthesis of Nap-FYp

[0057] A peptide gelling agent with the sequence Nap-Phe-Phe-Tyr(H2PO3)-Lys-OH was synthesized using a solid-phase peptide synthesizer. First, 2 g of 2-chlorotriphenylmethyl chloride resin was swollen in 20 mL of N,N-dimethylformamide (DMF) solution for 30 min. Then, the first amino acid, Fmoc-Tyr(H2PO3)-OH, was grafted onto the resin containing N,N-diisopropylethylamine (DIPEA, 800 μL) in DMF solution for 1 h. After washing three times with DMF to remove ungrafted amino acids, the resin was capped with a capping solution (DMF:methanol = 19:1) for 30 min. Next, the Fmoc protecting group was removed with a 20% piperidine-DMF solution for 30 min, followed by washing three times with DMF to remove residual piperidine. Next, 1-hydroxybenzotriazole (HOBt) / O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) / DIPEA was used as a coupling agent to couple the next amino acid, Fmoc-Lys(Boc)-OH (3.2 mmol), to the free amino group. Peptide growth was achieved by repeating these coupling and deprotection steps. Finally, after washing with DMF (5 times), isopropanol (5 times), and n-hexane (5 times), the synthesized peptide was cleaved from the resin with a 95% trifluoroacetic acid-dichloromethane solution for 30 min. The cleaved product was purified using a semi-preparative high-performance liquid chromatography (HPLC). Specifically, a water-acetonitrile solution containing 0.1% trifluoroacetic acid was used as the mobile phase eluent (from 60:40 to 0:100) to purify the impurity-containing peptide gelling factor (Nap-FYp). After obtaining the purified Nap-FYp, the acetonitrile in the purified Nap-FYp was removed using a rotary evaporator, and the Nap-FYp was dried in a lyophilizer for 24 hours to remove water from the purified peptide solution, finally obtaining a pure white powder of peptide gelling factor Nap-FYp.

[0058] (3) Synthesis of Nap-FYp-CPT

[0059] Weigh out 134 mg CPT-COOH (0.3 mM), 250 mg Nap-FYp (0.3 mM), 61 mg 1-hydroxy-7-azobenzotriazole (HOAt) (0.45 mM), and 171 mg 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (0.45 mM) and dissolve them in 2 mL of LDMF solution. Then, measure out 153 μL of LDIPEA and add it to the reaction solution. React in a 40°C water bath for 12 h. Figure 3As shown. The cleaved product was purified using semi-preparative high-performance liquid chromatography (HPLC). Specifically, a water-acetonitrile solution containing 0.1% trifluoroacetic acid was used as the mobile phase eluent (from 60:40 to 0:100) to purify Nap-FYp-CPT containing impurities. After obtaining purified Nap-FYp-CPT, acetonitrile was removed from the purified Nap-FYp-CPT using a rotary evaporator, and the product was then lyophilized for 24 hours to remove water from the purified peptide solution, finally yielding a pure yellow powder of Nap-FYp-CPT. The correctness of the Nap-FYp-CPT synthesis was further confirmed by mass spectrometry. Specifically, the molecular weight of Nap-FYp-CPT was 1281.3 m / z, and the hydrogenation result of 1282.3 m / z was consistent with the observed result, as shown in the figure. Figure 4 As shown, the correctness of the synthesis is proven.

[0060] Example 5: Chitosan-modified mesoporous silica nanoparticles loaded with the peptide prodrug Nap-FYp-CPT

[0061] 2 mg of chitosan-modified mesoporous silica nanoparticles were dispersed in 2 mL of dimethyl sulfoxide solution, and 10 mg of the peptide prodrug Nap-FYp-CPT was added. The reaction solution was incubated in a 37°C water bath for 12 h, and then washed three times by centrifugation with phosphate-buffered saline (PBS, pH 7.4) to obtain the mesoporous silica nanoparticles loaded with the peptide prodrug (Nap-FYp-CPT@MSN-CHI). Figure 1 As shown.

[0062] Example 6: Observation of the microstructure of Nap-FYp-CPT@MSN-CHI

[0063] Nap-FYp-CPT@MSN-CHI was diluted 10 times and dropped onto a carbon-coated copper grid, then air-dried overnight; the images were then observed and obtained using a JEM-2100 transmission electron microscope.

[0064] like Figure 2 As shown, the nanoparticles are about 120 nm in size and have clear mesoporous morphology.

[0065] Example 7: Experiment on the gelation of mesoporous silica nanoparticles mediated by both pH and alkaline phosphatase

[0066] Dissolve 2 mg Nap-FYp-CPT@MSN-CHI in 1 mL PBS solution (pH = 6.5), incubate for 24 h, then add alkaline phosphatase (50 U / mL) to the solution and incubate for another 24 h to observe the solution morphology.

[0067] like Figure 5As shown, the solution transforms into a stable and robust gel.

[0068] Example 8: CCK-8 assay for 4T1 cell viability

[0069] 4T1 cells were cultured in DMEM medium containing 10% FBS. 4T1 cells were seeded into 96-well plates (5000 cells / well). After 24 h, medium (Ctrl group) and Nap-FYp-CPT@MSN-CHI (0.12 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, 8 mg / mL) were added to the plates. After 48 h, the medium in each well was replaced with 100 μL DMEM medium containing 10 μL CCK-8 solution. After incubation in a cell culture incubator for 2 h, the absorbance (OD) at 450 nm was measured using a microplate reader; each experiment was performed at least three times.

[0070] like Figure 6 As shown, Nap-FYp-CPT@MSN-CHI exhibits concentration-dependent cytotoxicity. At a drug concentration of 2 mg / mL, the cell killing rate can reach approximately 50%.

[0071] Example 9: In vivo therapeutic efficacy evaluation of Nap-FYp-CPT@MSN-CHI

[0072] 1. Establishment of animal models

[0073] Four-week-old female BALB / c nude mice (16±2g) were selected; all experimental procedures complied with the provisions of the "Regulations on the Administration of Laboratory Animal Affairs in China"; in order to establish a tumor-bearing nude mouse model, 8×10 6 Four T1 cells (suspended in 100 μL PBS) were subcutaneously injected into the right back of each mouse.

[0074] 2. In vivo evaluation

[0075] To observe the retention of mesoporous silica nanoparticles at tumor sites, the fluorescent dye IR-820 was grafted onto the surface of the mesoporous silica nanoparticles. Once the tumor volume reached a certain size, nude mice were randomly divided into two groups: one group received intravenous injection of saline, and the other received injection of Nap-FYp-CPT@MSN-CHI grafted with IR-820 via tail vein. Tumor volume and mouse weight were measured daily during the treatment period. After treatment, the tumors were removed for volume analysis to evaluate the tumor inhibition effect.

[0076] like Figure 7 As shown, Nap-FYp-CPT@MSN-CHI remained at the tumor site for up to 7 days and significantly inhibited tumor growth.

Claims

1. A mesoporous silica nanoparticle, characterized in that, The nanoparticles are epoxy-modified, and their mesoporous channels are loaded with polypeptide precursor molecules grafted with chemotherapeutic drug molecules, and their outer layer is coated with an acid-sensitive polymer layer; the polypeptide precursor molecules are selected from Nap-Phe-Phe-Tyr(H2PO3)-Lys-OH, wherein the Lys amino acid side is grafted with chemotherapeutic drug molecules; the acid-sensitive polymer is selected from chitosan.

2. The mesoporous silica nanoparticles according to claim 1, characterized in that, The chemotherapy drug molecule is selected from one or more of paclitaxel, camptothecin, and dobirubin.

3. The mesoporous silica nanoparticles according to claim 1, characterized in that, The nanoparticles have a particle size of 100~120 nm and a PDI < 0.

2.

4. A method for preparing mesoporous silica nanoparticles according to claim 1, characterized in that, Includes the following steps: (1) Preparation of epoxy-modified mesoporous silica nanoparticles; (2) The nanoparticles prepared in step (1) are coated with an acid-sensitive polymer; (3) The nanoparticles prepared in step (2) are loaded with chemotherapeutic drug molecules and polypeptide precursor molecules in the mesoporous channels.

5. A pharmaceutical composition, characterized in that, It comprises the mesoporous silica nanoparticles of claim 1 and a pharmaceutically acceptable carrier.

6. A molecular probe, characterized in that, It is obtained by loading fluorescent dye onto mesoporous silica nanoparticles as described in claim 1.

7. The use of the mesoporous silica nanoparticles of claim 1, the pharmaceutical composition of claim 5, or the molecular probe of claim 6 in the preparation of a medicament for treating tumors.

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