CN110538329A - Preparation method of pH-responsive mesoporous silica nano-drug carrier for three-in-one therapy - Google Patents
Preparation method of pH-responsive mesoporous silica nano-drug carrier for three-in-one therapy Download PDFInfo
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Abstract
A preparation method of a pH response mesoporous silica nano-drug carrier for a three-in-one therapy belongs to the field of drug carrier preparation, can solve the problem that the difficulty of the existing combined therapy realized on the same nano-platform by a simple means is high, and comprises mesoporous silica nanoparticles loaded with anti-cancer drugs, a polydopamine shell layer complexed with divalent manganese ions and a photosensitizer. After the nano-carrier is taken up by tumor cells, the poly-dopamine shell is degraded in a slightly acidic environment to trigger the release of the pH-responsive anticancer drug and the photosensitizer; the methylene blue reacts with dissolved oxygen in cells under the radiation of 635nm laser to generate highly toxic hydroxyl free radicals, and meanwhile, the complexed divalent manganese ions on the polydopamine and hydrogen peroxide in the cells generate Fenton-like reaction to further promote the generation of the highly toxic hydroxyl free radicals, so that the three-in-one treatment process of photodynamic therapy/chemodynamic therapy/chemotherapy is realized.
Description
Technical Field
The invention belongs to the technical field of preparation of drug carriers, and particularly relates to a preparation method of a pH response mesoporous silica nano drug carrier for photodynamic therapy/chemodynamic therapy/chemotherapy.
Background
Cancer seriously threatens the life and health of human beings. Currently, chemotherapy remains the most common means of treating cancer. However, chemotherapy drugs often have systemic toxicity, causing serious adverse effects. Therefore, it is important to develop a new formulation with specific environment for responding to tumor. With the development of nanotechnology, the target drug carrier based on the nanometer material provides a new scheme for realizing tumor-specific drug delivery. Moreover, nano-drug carriers often also improve the bioavailability, permeability and stability of anticancer drugs compared to traditional pharmaceutical dosage forms, which further ensures a sustained and controlled drug delivery.
Among a series of studied nano materials, mesoporous silica is an ideal candidate material due to the characteristics of good biocompatibility, high specific surface area, easy surface modification and the like. Since mesoporous silica was first used in the field of drug carriers in 2003, further development of novel carriers based on mesoporous silica has received extensive attention from researchers. In the synthesis process of early silica-based drug carriers, the surface of the material is not modified, and the premature release of the drug and the toxic and side effects are still caused, so that the further development of the carriers is limited. One possible solution to this bottleneck is to modify the silicon surface with molecular switches to achieve release under specific conditions. The polydopamine is a high molecular polymer formed by oxidizing and self-polymerizing dopamine, is easy to biodegrade due to the unstable structure of the polydopamine under an acidic condition, and becomes an ideal pH response molecular switch. Based on the research personnel, a series of nano drug-carrying platforms taking polydopamine as functional macromolecules are also constructed, and certain effects are achieved. However, tumor development often involves numerous signaling pathways, and the efflux proteins on the surface of the tumor impair the therapeutic effect of chemotherapeutic drugs. Therefore, the construction of a nano platform with the synergistic effect of multiple therapies is the mainstream of the current research.
Reactive Oxygen Species (ROS), a class of free radicals with very strong oxidative activity, are capable of causing irreversible damage to the molecular structure of DNA and proteins in cells, resulting in apoptosis of cells. Two of the most commonly used means for intracellular generation of reactive oxygen species are photodynamic therapy (PDT) and chemodynamic therapy (CDT). For PDT process, the photosensitizer reacts with dissolved oxygen in cells photochemically under the excitation of an excitation light source to generate hydroxyl radicals; the CDT process delivers a fenton reagent having the ability to generate hydroxyl radicals into tumor cells, and then reacts with excess hydrogen peroxide in the cells to generate radicals. Because the reaction materials involved in PDT and CDT processes are not interfered with each other, PDT and CDT are combined to form an enhanced active oxygen mediated treatment process, so that the enhanced active oxygen mediated treatment process becomes an attractive novel tumor combination therapy. To our knowledge, however, strategies for achieving chemotherapy, chemodynamic therapy and photodynamic therapy by simple means on the same nano-platform remain a significant challenge.
Disclosure of Invention
The invention provides a preparation method of a pH response mesoporous silica nano-drug carrier for photodynamic therapy/chemodynamic therapy/chemotherapy, aiming at the problem of great difficulty in realizing chemotherapy, chemodynamic therapy and photodynamic therapy combination therapy on the same nano-platform by a simple means.
The invention designs and synthesizes a drug carrier based on mesoporous silica nano material by combining the advantages of mesoporous silica and polydopamine. Doxorubicin is loaded by mesoporous pore channels, manganese ions of a Fenton reagent are complexed by a catechol structure in polydopamine, and photosensitizer methylene blue is loaded on the surface of the carrier through pi-pi accumulation to realize integration of chemotherapy, chemical dynamic therapy and photodynamic therapy, so that a potential solution idea is provided for developing a nano carrier with the synergistic effect of multiple therapies.
The invention adopts the following technical scheme:
A preparation method of a pH response mesoporous silicon dioxide nano-drug carrier for a three-in-one therapy comprises the following steps:
Firstly, preparing Mesoporous Silica Nanoparticles (MSN) by a sol-gel method;
Secondly, dispersing the obtained mesoporous silica nanoparticles in a Tris-HCl buffer solution containing dopamine, and reacting for 12 hours in a dark place to obtain polydopamine modified mesoporous silica nanoparticles (MP);
Dispersing the obtained poly-dopamine modified silica nanoparticles in an ethanol solution containing acetic acid metal ions, and stirring to obtain poly-dopamine metal ion complex modified mesoporous silica nanoparticles (MPM);
And fourthly, dissolving the photosensitizer in water to obtain a mixed solution, dispersing the obtained mesoporous silica nanoparticles modified by the polydopamine metal ion complex into the mixed solution, stirring the mixed solution at room temperature in a dark place for 24 hours, after the reaction is finished, centrifugally washing, and drying in vacuum to obtain the photosensitizer-loaded nanoparticles (MPMB).
The method for preparing the mesoporous silica nano particles by the sol-gel method in the first step comprises the following steps:
And (2) catalyzing the precursor by using a surfactant CTAB (cetyl trimethyl ammonium bromide) as a precursor and TEOS (tetraethyl orthosilicate) as a silicon source under an alkaline condition to obtain solid silica nanoparticles, and dispersing the solid silica nanoparticles into a concentrated hydrochloric acid and ethanol system or ammonium nitrate-ethanol mixed solution to reflux for 24 hours to remove the precursor to obtain the mesoporous silica nanoparticles.
in the second step, the pH value of the Tris-HCl buffer solution is 8.5, the concentration of the Tris-HCl buffer solution is 10mM, and the mass ratio of the dopamine to the mesoporous silica nano particles is 1: 1-2.
the metal ion in the third step includes any one of manganese ion, ferrous ion, ferric ion and cupric ion.
In the third step, the ethanol solution of the metal ions is a manganese acetate ethanol solution with the concentration of 10-20 mM.
In the fourth step, the photosensitizer comprises any one of Methylene Blue (MB), indocyanine green, bengal red, MC-540 and chlorin e6, and the mass ratio of the photosensitizer to the mesoporous silica nanoparticles modified by the polydopamine metal ion complex is 0.25-0.5: 1.
the principle of the invention is as follows:
After the nano-carrier is taken up by tumor cells, the polydopamine shell is degraded in a slightly acidic environment, so that the release of the pH-responsive anticancer drug and the photosensitizer is triggered; the methylene blue can react with dissolved oxygen in cells to generate highly toxic hydroxyl radicals under the radiation of 635nm laser, and meanwhile, the complexed divalent manganese ions on the polydopamine and hydrogen peroxide in the cells generate Fenton-like reaction to further promote the generation of the highly toxic hydroxyl radicals, thereby realizing the three-in-one treatment process of photodynamic therapy/chemodynamic therapy/chemotherapy. Moreover, the manganese ions can be used as a contrast agent for magnetic resonance imaging to remarkably improve imaging precision. The designed and synthesized drug carrier integrates the three therapies into the same nano platform, promotes the anti-tumor effect and provides a potential feasible path for realizing the multi-therapy cooperative treatment of tumors.
the invention has the following beneficial effects:
1. The pH response mesoporous silica nano-drug carrier for the photodynamic therapy/chemodynamic therapy/chemotherapy has obvious pH triggered drug release property and the effect of improving the tumor cytotoxicity by combining chemotherapy/photodynamic therapy/chemodynamic therapy.
2. The manganese ions are used as Fenton reagents, and the manganese ions are directly combined with the catechol structure in the polydopamine, so that the defects of complex carrier synthesis, low yield and potential damage of multistep reaction to a medicine structure caused by the need of modifying manganese dioxide are avoided. Realizes the rapid, mild and stable modification process.
3. The polydopamine has a larger benzene ring system, so that the polydopamine has the function of pi-pi accumulation on a plurality of photosensitizers, so that the polydopamine has the potential of being applied to different photosensitizer-mediated photodynamic therapy, and the potential application range is expanded.
Drawings
Fig. 1 is a transmission electron microscope image of mesoporous silica nanoparticles M prepared in example 1 of the present invention.
Fig. 2 is a transmission electron microscope image of the pH-responsive mesoporous silica nano-drug carrier MPMB prepared in example 1 of the present invention.
Fig. 3 is an XPS composition analysis diagram of the mesoporous silica nanoparticle MSN prepared in example 1 of the present invention.
fig. 4 is an XPS composition analysis diagram of the poly-dopamine-modified mesoporous silica nanoparticle MP prepared in example 1 of the present invention.
Fig. 5 is an XPS composition analysis diagram of the pH-responsive mesoporous silica nano-drug carrier MPMB prepared in example 1 of the present invention.
FIG. 6 is a graph showing the cumulative release of DOX at different pH values in DOX/MPMB nanocarriers prepared in example 1 of the present invention.
FIG. 7 is a graph showing the cumulative release of MB at different pH's in DOX/MPMB nanocarriers prepared in example 1 of the invention.
FIG. 8 is a diagram of free radical generation in a cell under a confocal laser scanning microscope, in which: A-C: culturing SMMC-7721 cells by using nano-carriers without photosensitizer, metal ions and medicine; D-F: treating SMMC-7721 cells with nanoparticles containing only methylene blue; G-I: treating SMMC-7721 cells with a pharmaceutical carrier containing only manganese ions; J-L: SMMC-7721 cells were treated with MPMB, a pharmaceutical vehicle containing a photosensitizer and manganese ions. Culturing time: 4 hours; DCFH-DA concentration: 10 mu M; particle concentration: 50 mu g/mL; a scale: 20 μm.
FIG. 9 is a graph of fluorescence images of cell drug delivery at different culture times for DOX/MPMB prepared according to example 1 of the present invention, wherein the concentration of nano-particles is 50 μ g/mL; a scale: 20 μm.
FIG. 10 is a graph of fluorescence images of drug delivery in different concentrations of nanoparticles of DOX/MPMB prepared in example 1 of the present invention, wherein the incubation time is 4h, and the scale: 20 μm.
FIG. 11 is a graph showing the cytotoxicity of DOX/MPMB prepared in example 1 of the present invention on tumor cells under different conditions, wherein the culture time: and (5) 24 h.
Detailed Description
Example 1
(1) 240 mL of water, 0.5 g of CTAB surfactant and 1.7 mL of 2M sodium hydroxide are added into a 500 mL volumetric flask, the mixture is slowly stirred and heated to 80 ℃, the rotation speed is increased, the mixture is vigorously stirred for 30 minutes, then 2.5 mL of TEOS is dropwise added, and a white suspension is obtained after reaction for two hours. Centrifuging, washing with ethanol for 2 times, re-dispersing in a mixed system of 140 mL of anhydrous ethanol and 6 mL of concentrated hydrochloric acid, refluxing for 24 hours, centrifuging, washing, and vacuum drying to obtain fine white powder to obtain Mesoporous Silica Nanoparticles (MSN).
(2) And (3) ultrasonically dispersing 200mg of MSN into 100 mL of Tris-HCl, adding 100mg of dopamine, and stirring for 12 hours in a dark place to obtain the poly-dopamine modified silicon dioxide nano particle MP. The color of the system changes to milky white, light pink, gray black and black in the reaction process.
(3) Weighing 200 mgMP solid, and ultrasonically dispersing in 20 mL absolute ethyl alcohol; manganese acetate was dissolved in 30 mL of absolute ethanol to prepare a 10mM manganese acetate solution, and the two solutions were mixed and stirred at room temperature for 1 hour. Centrifuging and drying in vacuum to obtain black solid MPM.
(4) 100mg of MPM were weighed, dispersed in water, and 25 mg of methylene blue was added. The two are mixed and stirred for 24 hours, and the black solid powder MPMB is obtained after centrifugal washing and vacuum drying.
The method for loading anticancer drug adriamycin (DOX) serving as a model drug in the mesoporous silica nanoparticles comprises the following steps: dissolving adriamycin in PBS buffer solution, adding mesoporous silica nanoparticles, stirring for 24h in the dark, centrifuging and washing, and performing subsequent surface modification steps such as the steps (2), (3) and (4). The mass ratio of the adriamycin to the mesoporous silica nano particles is 0.25: 1; the loading medium is PBS 7.4, 10 mM; the loading time was 24 hours.
example 2
(1) 240 mL of water, 0.5 g of CTAB surfactant and 1.7 mL of 2M sodium hydroxide are added into a 500 mL volumetric flask, the mixture is slowly stirred and heated to 80 ℃, the rotation speed is increased, the mixture is vigorously stirred for 30 minutes, then 2.5 mL of TEOS is dropwise added, and a white suspension is obtained after reaction for two hours. Centrifuging, washing with ethanol for 2 times, re-dispersing in 140 mL of 10 mg/mL ammonium nitrate-ethanol mixed solution, refluxing for 24 hours, centrifuging, washing, and vacuum drying to obtain fine white powder to obtain Mesoporous Silica Nanoparticles (MSN).
(2) and (3) ultrasonically dispersing 200mg of MSN in 100 mL of Tris-HCl, adding 200mg of dopamine, and stirring for 12 hours in a dark place to obtain the poly-dopamine modified silicon dioxide nano particle MP.
(3) weighing 200 mgMP solid, and ultrasonically dispersing in 20 mL absolute ethyl alcohol; manganese acetate was dissolved in 30 mL of absolute ethanol to prepare a 20mM manganese acetate solution, and the two solutions were mixed and stirred at room temperature for 1 hour. Centrifuging and drying in vacuum to obtain black solid MPM.
(4) 100mg of MPM were weighed, dispersed in water, and 50 mg of methylene blue was added. The two are mixed and stirred for 24 hours, and the black solid powder MPMB is obtained after centrifugal washing and vacuum drying.
The method for loading anticancer drug adriamycin (DOX) serving as a model drug in the mesoporous silica nanoparticles comprises the following steps: dissolving adriamycin in PBS buffer solution, adding mesoporous silica nanoparticles, stirring for 24h in the dark, centrifuging and washing, and performing subsequent surface modification steps such as the steps (2), (3) and (4). The mass ratio of the adriamycin to the mesoporous silica nano particles is 0.5: 1; the loading medium is PBS 7.4, 10 mM; the loading time was 24 hours.
the used medicine adriamycin can be epirubicin, camptothecin, taxol, curcumin and fluorouracil.
Use of MPMB for photodynamic therapy/chemodynamic therapy/chemotherapy combination therapy.
Example 1 characterization test for drug carriers:
(1) transmission Electron Microscopy (TEM) results
as shown in fig. 1, TEM test results show that: the mesoporous silica nano particles are approximately spherical, have the particle size of about 100 nm and have an ordered pore channel structure.
As shown in fig. 2, after surface modification, a distinct polymer layer appears on the surface of the silica nanoparticles, and the pore channel structure is blurred.
(2) X-ray photoelectron spectroscopy (XPS) results
As shown in fig. 3, TEM test results show that: only the Si 2p, Si 2s and O1 s peaks were observed in the XPS survey of MSN.
As shown in fig. 5, MPM observed N1 s peak from nitrogen element and Mn 2p peak from manganese element in dopamine after modification with polydopamine and manganese.
(3) DOX/MPMB nano-carrier release results
To examine the release of DOX, 3 mg of the above drug-loaded complex was released at 37 ℃ in 10 mL of different release media (1: pH 7.4; 2: pH 6.3; 3: pH 5.3). 2 mL of solution was withdrawn at intervals while fresh medium was replenished. The effect of release of DOX (480 nm) was studied using uv-vis spectroscopy.
To examine release of MB, 3 mg of the above drug-loaded complex was released at 37 ℃ in 10 mL of different release media (1: pH 7.4; 2: pH 5.3). 2 mL of solution was withdrawn at intervals while fresh medium was replenished. The release effect on MB (635 nm) was studied using uv-vis spectrometer.
As shown in fig. 6 and 7, the drug release showed 15.0% and 15.5% release of DOX and MB, respectively, at pH 7.4, indicating that the drug release was low in physiological environments. The cumulative release of DOX and MB gradually increased with decreasing pH, over that released under physiological conditions, indicating that the vehicle had good release response.
(4) a series of human hepatoma cells SMMC-7721 were cultured in sterile DMEM under the following cell assay conditions:
1. Adding MPMB carrier without methylene blue, adriamycin and bivalent manganese ions, and taking the concentration of the MPMB carrier as a control group at 50 mug/mL;
2. Group 1 was added MPMB vehicle containing only MB at a concentration of 50. mu.g/mL, irradiated with 635nm laser (10 min, 0.1W/cm 2)
3. Group 2 was added MPMB vehicle containing only manganese ions at a concentration of 50. mu.g/mL, irradiated with a 635nm laser (10 min, 0.1W/cm 2);
4. group 3 was added MPMB vehicle containing MB and manganese ions at a concentration of 50. mu.g/mL, irradiated with a 635nm laser (10 min, 0.1W/cm 2);
After 4 hours of incubation, DCFH-DA was used as an active oxygen indicator and observed with a confocal laser microscope (excitation: 488 nm, collection emission: 505 and 560 nm; microscope model: Zeiss LSM 880)
The results show that group 3 has the strongest reactive oxygen species generating capacity compared to the remaining groups, indicating that the MPMB carrier has therapeutic potential for photodynamic and chemokinetic process-mediated reactive oxygen species. See fig. 8.
(5) A series of human liver cancer cells SMMC-7721 are cultured by using sterile DMEM, then 7721 cells are cultured by using DMEM to prepare DOX/MPPF nano particles with the concentration of 50 mu g/mL, after 1, 2 and 4 hours of culture, a cell nucleus fluorescent stain Hoechst 33258 with the volume of 1/10 culture solution is added, after the cells are thoroughly washed by using sterile PBS, the culture dish is placed into a laser confocal microscope for detection. (nuclear stain excitation wavelength: 305 nm, collection emission segment: 385-435 nm, blue light channel; doxorubicin excitation wavelength: 488 nm, collection emission segment: 560-620 nm, orange light channel; methylene blue excitation wavelength: 633 nm, collection emission segment: 630-660 nm, red light channel; microscope model: Zeiss LSM 880). The result shows that the methylene blue and the adriamycin loaded by the carrier are smoothly taken up by the tumor cells, and the two medicines are integrally distributed in the cytoplasm range, which indicates that the carrier can smoothly enter the cells and further exert the treatment effect. As shown in fig. 9.
(6) A series of human liver cancer cells SMMC-7721 are cultured by using sterile DMEM, then different DOX/MPPF nano particles are prepared by using DMEM to culture 7721 cells (25, 50 and 100 mu g/mL), after 4 hours of culture, a cell nucleus fluorescent stain Hoechst 33258 with the volume of 1/10 culture solution is added, after the cells are thoroughly washed by using sterile PBS, the culture dish is placed into a laser confocal microscope for detection. (nuclear stain excitation wavelength: 305 nm, collection emission segment: 385-435 nm, blue light channel; doxorubicin excitation wavelength: 488 nm, collection emission segment: 560-620 nm, orange light channel; methylene blue excitation wavelength: 633 nm, collection emission segment: 630-660 nm, red light channel; microscope model: Zeiss LSM 880). The results show that the fluorescence intensity of DOX and MB increases gradually with increasing carrier concentration and the two drugs remain predominantly distributed in the cytoplasmic range. With reference to fig. 5, the time-dependent and concentration-dependent uptake of nanoparticles by cells is illustrated. As shown in fig. 10.
(7) A series of human liver cancer cells SMMC-7721 are cultured by sterile DMEM, and cytotoxicity tests are carried out on drug carriers with different compositions and different concentrations by adopting an MTT method. The results show that the DOX/MPMB nano-particles show the strongest cell killing capability compared with the free drug, and the designed and synthesized drug carrier has the potential for chemotherapy/photodynamic therapy/chemodynamic therapy synergistic treatment. As shown in fig. 11.
Claims (6)
1. A preparation method of a pH response mesoporous silicon dioxide nano-drug carrier for a three-in-one therapy is characterized by comprising the following steps: the method comprises the following steps:
firstly, preparing mesoporous silica nano particles MSN by a sol-gel method;
Secondly, dispersing the obtained mesoporous silica nanoparticles in a Tris-HCl buffer solution containing dopamine, and reacting for 12 hours in a dark place to obtain polydopamine-modified mesoporous silica nanoparticles MP;
Dispersing the obtained poly-dopamine modified silica nanoparticles in an ethanol solution containing acetic acid metal ions, and stirring to obtain poly-dopamine metal ion complex modified mesoporous silica nanoparticles MPM;
And fourthly, dissolving the photosensitizer in water to obtain a mixed solution, dispersing the obtained mesoporous silica nanoparticles modified by the polydopamine metal ion complex into the mixed solution, stirring the mixed solution at room temperature in a dark place for 24 hours, after the reaction is finished, centrifugally washing, and drying in vacuum to obtain the photosensitizer-loaded nanoparticle MPMB.
2. the method for preparing pH-responsive mesoporous silica nano-drug carrier for three-in-one therapy according to claim 1, characterized in that: the method for preparing the mesoporous silica nano particles by the sol-gel method in the first step comprises the following steps:
And (2) catalyzing the precursor by using a surfactant CTAB (cetyl trimethyl ammonium bromide) as a precursor and TEOS (tetraethyl orthosilicate) as a silicon source under an alkaline condition to obtain solid silica nanoparticles, and dispersing the solid silica nanoparticles into a concentrated hydrochloric acid and ethanol system or ammonium nitrate-ethanol mixed solution to reflux for 24 hours to remove the precursor to obtain the mesoporous silica nanoparticles.
3. The method for preparing pH-responsive mesoporous silica nano-drug carrier for three-in-one therapy according to claim 1, characterized in that: in the second step, the pH value of the Tris-HCl buffer solution is 8.5, the concentration of the Tris-HCl buffer solution is 10mM, and the mass ratio of the dopamine to the mesoporous silica nano particles is 1: 1-2.
4. The method for preparing pH-responsive mesoporous silica nano-drug carrier for three-in-one therapy according to claim 1, characterized in that: the metal ion in the third step includes any one of manganese ion, ferrous ion, ferric ion and cupric ion.
5. The method for preparing pH-responsive mesoporous silica nano-drug carrier for three-in-one therapy according to claim 1, characterized in that: in the third step, the ethanol solution of the metal ions is a manganese acetate ethanol solution with the concentration of 10-20 mM.
6. The method for preparing pH-responsive mesoporous silica nano-drug carrier for three-in-one therapy according to claim 1, characterized in that: in the fourth step, the photosensitizer comprises any one of methylene blue MB, indocyanine green, bengal red, MC-540 and chlorin e6, and the mass ratio of the photosensitizer to the mesoporous silica nanoparticles modified by the polydopamine metal ion complex is 0.25-0.5: 1.
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