CN114344464B - Self-carrying oxygen mitochondria-targeted photodynamic therapy nanoplatform, preparation method and application - Google Patents

Abstract

The invention belongs to the technical field of photodynamic therapy, and discloses a self-oxygen-carrying mitochondrial targeting photodynamic therapy nano platform, a preparation method and application. The preparation method comprises the steps of regulating and controlling the growth structure of the organic silicon oxide by a two-step silicon source adding method to obtain eccentric organic silicon oxide and obtaining eccentric mesoporous organic silicon oxide by a hydrothermal etching method, carrying out functional modification on the surface of the organic silicon oxide to obtain mitochondrial targeting and photosensitizer, and loading perfluorocarbon with low boiling point and self oxygen carrying characteristics in a cavity. The prepared photodynamic therapy nano platform has a large internal cavity and a mesoporous pore canal, can efficiently load oxygen-carrying perfluorocarbon molecules to target mitochondria, and has high singlet oxygen generation characteristic and cancer cell killing characteristic after being irradiated by laser with specific wavelength. The preparation method is simple and easy to obtain, and the obtained product has high yield and great application potential in the fields of tumor photodynamic therapy and the like.

Description

Self-carrying oxygen mitochondria-targeted photodynamic therapy nanoplatform, preparation method and application

Technical field

The invention belongs to the technical field of photodynamic therapy. Specifically, it relates to a photodynamic nano-platform with self-carrying oxygen properties and mitochondrial targeting properties and its preparation method and application.

Background technique

Photodynamic therapy uses a photosensitizer to convert oxygen into cytotoxic reactive oxygen species under laser irradiation with a suitable wavelength, thereby killing tumor cells. Compared with traditional tumor treatment methods, photodynamic therapy has the advantages of less trauma, good reproducibility, high spatial selectivity, and few side effects. Oxygen, as one of the three major elements of photodynamic therapy, is closely related to the therapeutic effect. Hypoxia is common in solid tumors and oxygen consumption during photodynamic therapy often forms a vicious cycle, which is not conducive to adequate tumor treatment. Effectively increasing the oxygen supply to the tumor site can improve the effect of photodynamic therapy. In recent years, photodynamic therapy based on nanomaterials has received more and more attention. The delivery of photosensitizers by nanomaterials can not only increase their water solubility and cellular uptake, but also utilize the various properties of the nanoparticles themselves or couple them with other functionalities. Molecules to achieve photodynamic therapy combined with oxygen supply, tumor targeting, etc.

Mesoporous organic silica is a type of mesoporous material whose structure is evenly doped with organic and inorganic components. They have regular and ordered mesopores, uniform and adjustable shape and size, large specific surface area and easy-to-modify surface properties. , which can efficiently load and deliver drugs, genes, imaging molecules, etc. to form a multifunctional whole. In addition, mesoporous organic silica also has good biosafety and biodegradability, laying the foundation for its application in the field of biomedicine. However, due to the single role of organic silica nanoparticles as nanodrug carriers, it is difficult to achieve the effect of treating tumors. Therefore, by combining the three characteristics of loading self-oxygen-carrying functional molecules, mitochondrial targeting and photodynamic therapy, mesoporous organic oxidation Silicon nanomaterials are more widely studied for biomedical applications, but many new applications remain to be explored.

Contents of the invention

The technical problem to be solved by the present invention is to provide a preparation method for an eccentric hollow mesoporous organic silica nanoparticle oxygen delivery platform and loading carboxytriphenylphosphorus (CTPP) and photosensitizer Ce6. The nanoplatform achieved large-scale aggregation of mitochondria and enhanced intracellular singlet oxygen production, further improving the killing efficiency of Ce6 cells. Based on the basic characteristics of mesoporous organic silica and photodynamic therapy, the present invention provides a nano-delivery platform of eccentric hollow mesoporous organic silica and a preparation method loaded with self-carrying oxygen molecules, modified carboxytriphenylphosphine and photosensitizer Ce6, It increases the oxygen content in the tumor while targeting mitochondria, further enhancing the effect of photodynamic therapy.

The present invention is realized through the following technical solutions:

In the first aspect, the present invention provides a self-carrying oxygen-carrying mitochondria-targeted photodynamic therapy nano-platform, which includes eccentric hollow mesoporous organic silica, mitochondrial targeting molecules modified on the surface of the eccentric hollow mesoporous organic silica, and a photosensitizer. and self-carrying oxygen perfluorocarbon molecules loaded in the eccentric cavity of eccentric hollow mesoporous organic silica. Among them, the eccentric hollow structure helps the gas in the cavity to vaporize and generate bubbles to push the particles to move, achieving the movement effect of the motor.

Further, the eccentric cavity size of the eccentric hollow mesoporous organic silica is 150-300 nm.

Further, the eccentric cavity surface of the eccentric hollow mesoporous organic silica has an ultrathin mesoporous shell layer.

Further, the eccentric hollow mesoporous organic silica has uniform mesoporous channels.

Further, the mitochondrial targeting molecule is carboxytriphenylphosphonium (CTPP).

Further, the photosensitizer is chlorin e6 (Ce6).

Further, the oxygen-carrying perfluorocarbon molecule is tetradecafluorohexane.

In a second aspect, the present invention provides a method for preparing a self-carrying oxygen mitochondria-targeted photodynamic therapy nanoplatform, which includes: using an inorganic silicon source to synthesize size-adjustable mesoporous inorganic silica, and using an organic silicon source to coat the Mesoporous inorganic silica, and then use hydrothermal method to remove the inorganic silica to form eccentric hollow mesoporous organic silica, and modify the surface of the eccentric hollow mesoporous organic silica through amination to jointly modify mitochondrial targeting molecules and photosensitizers, and By loading self-carrying oxygen perfluorocarbon molecules into the eccentric cavity of eccentric hollow mesoporous organic silica, a self-carrying oxygen mitochondria-targeted photodynamic therapy nanoplatform was obtained.

Furthermore, in the process of synthesizing eccentric hollow mesoporous organic silica, an organic silicon source is grown heterogeneously on the surface of mesoporous inorganic silica. The eccentricity is obtained under the conditions of 35°C, 500 rpm, and the ratio of ammonia water and organic silicon source is 1:100-25. Hollow mesoporous organic silica.

Further, the inorganic silicon source is TEOS, the organic silicon source is BTSE, and the volume ratio of the two is 1:0.05-0.5.

Furthermore, during the synthesis of eccentric hollow mesoporous organic silica, inorganic silica was removed by hydrothermal method in a water bath at 80°C for 12 hours to form eccentric hollow mesoporous organic silica.

Furthermore, the eccentric hollow mesoporous organic silica modified with mitochondrial targeting molecules and photosensitizers was mixed with self-carrying oxygen perfluorocarbon molecules under vacuum conditions, and ultrasonicated in an ice bath to obtain self-carrying oxygen mitochondria-targeted photodynamic force. Therapeutic Nanoplatforms.

Specifically, a method for preparing a self-carrying oxygen mitochondria-targeted photodynamic therapy nanoplatform includes the following steps:

(1) Synthesis of inorganic silica nanoparticles (MSNs): 170 ml cetyltrimethylammonium bromide (CTAB, 6 mM) was added to a mixture of 75 ml ethanol and 0.1 ml ammonia water, at 35°C, rotating speed 500 Add 0.2 ml of tetraethyl orthosilicate (TEOS) with stirring at rpm, adjust the temperature to 60°C, react for 24 to 48 hours, centrifuge, and wash with absolute ethanol three times to obtain inorganic silicon oxide nanoparticles;

(2) Synthesis of eccentric hollow mesoporous organic silicas (JMONs): Take 3.5 mg of inorganic silica nanoparticles, centrifuge them in a centrifuge tube, and disperse them in 0.5 ml ethanol, 8.5 ml deionized water, 0.015 g CTAB, and 0.2~0.7 ml ammonia water. To the mixture, stir for 30 minutes at 35°C and 500 rpm, add 0.01~0.1 ml 1,2-bis(triethoxysilyl)ethane (BTSE), continue the reaction for 3 hours, centrifuge, and add ethanol Wash three times and once with water. Place the sample in an 80°C water bath for 12 hours, centrifuge, and wash twice with ethanol. Place it in a mixture of 200 ml ethanol and 0.4 ml concentrated hydrochloric acid, and react at 60°C and 1100 rpm. Repeat three times. , the reaction times are 3 hours, 12 hours, and 3 hours respectively. The final product is washed three times with ethanol and dissolved in 30 ml of ethanol to obtain eccentric hollow mesoporous organic silica nanoparticles;

(3) Modification of Ce6 and CTPP: First, mix 1 ml of carboxytriphenylphosphine CTPP (20 mg/ml) and 1 ml of photosensitizer Ce6 (20 mg/ml) with 0.5 ml of bis[3-(triethoxysilyl) )propyl]tetrasulfide EDC (20 mg/ml in N,N-dimethylformamide (DMF)) and 0.5 ml of N-hydroxysuccinimide NHS (20 mg/ml in DMF) were mixed. The mixture was shaken at room temperature for 3 hours to activate the carboxyl groups. 1 ml of nanoparticles was then dispersed into the carboxyl-activated CTPP solution. After reacting for 12 hours, centrifuge and wash three times with water, then disperse into carboxyl-activated Ce6, react for 12 hours, and centrifuge and wash three times with water to obtain stable surface functionally modified JMONs-Ce6&CTPP.

(4) Synthesis of self-carrying oxygen eccentric hollow mesoporous organic silica nanoparticles (JMONs- Ce6&CTPP@PFC): Centrifuge 1 mg of surface-modified JMONs- Ce6&CTPP at 10000 rpm, wash with water twice, and quickly add 0.1 ml after vacuuming PFC solution was sonicated in an ice bath for 2 minutes to obtain JMONs- Ce6&CTPP@PFC.

In a third aspect, the present invention also provides the application of the aforementioned self-carrying oxygen mitochondria-targeted photodynamic therapy nano-platform in the preparation of anti-tumor drugs.

Beneficial effects:

1. The photodynamic therapy nano-platform provided by the present invention has a large internal cavity and mesoporous channels, can efficiently load perfluorocarbons and has efficient release characteristics, which is conducive to the transportation of large amounts of oxygen;

2. Preparation of eccentric hollow mesoporous organic silica nanoparticles through a simple hydrothermal method, which requires low equipment requirements, low cost, and is environmentally friendly; the preparation method provided by the invention is simple and easy to obtain, and the yield of the product obtained is high, It has huge application potential in fields such as tumor photodynamic therapy;

3. Materials with self-carrying oxygen properties have a simpler design and implementation of tumor oxygenation, and have obvious singlet oxygen production compared to blank experiments;

4. Eccentric hollow mesoporous silicone nanoparticles combine mitochondrial targeting function to improve mitochondrial targeting ability, intracellular singlet oxygen production efficiency and photodynamic therapy effect.

Description of drawings

Figure 1 is the working principle of the self-carrying oxygen mitochondria-targeted photodynamic therapy nano-platform in an embodiment of the present invention;

Figure 2 is the structural formula of the photosensitizer Ce6 and carboxytriphenylphosphonium (CTPP);

Figure 3 is a transmission electron microscope (TEM), scanning electron microscope (SEM) and hydration kinetic size characterization diagram of the inorganic silicon oxide nanoparticles prepared in Example 1 of the present invention;

Figure 4 shows the transmission electron microscope (TEM), scanning electron microscope (SEM), water and kinetic size, element distribution, and Fourier transform infrared spectrum of the eccentric hollow mesoporous organic silica nanoparticles prepared in Example 1 of the present invention. , Nitrogen adsorption and desorption curve image;

Figure 5 is an image of the UV-visible absorption spectrum, hydration kinetic size, surface potential and element distribution of the eccentric hollow mesoporous organic silica nanoparticles modified with Ce6 and CTPP prepared in Example 1 of the present invention;

Figure 6 is a cytocompatibility diagram of the surface-modified eccentric hollow mesoporous organic silica nanoparticles produced in Example 1 of the present invention;

Figure 7 is a comparison diagram of the intracellular and extracellular singlet oxygen production of prepared eccentric hollow mesoporous organic silica nanoparticles loaded with perfluorocarbon and surface modified in Example 1 of the present invention with or without laser irradiation;

Figure 8 is a comparative diagram of targeted photodynamic therapy of eccentric hollow mesoporous organic silica nanoparticles loaded with perfluorocarbon and surface modified prepared in Example 1 of the present invention.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. The following examples are only used to more clearly illustrate the technical solutions of the present invention, but cannot be used to limit the scope of the present invention.

With reference to Figures 1 and 2, the preparation method of an eccentric hollow mesoporous organic silica nanoparticle delivery platform loaded with perfluorocarbon, CTPP and photosensitizer Ce6 is introduced in detail.

Example 1:

1. Synthesis of inorganic silica nanoparticles (MSNs):

First, prepare a 6 mM aqueous solution of cetyltrimethylammonium bromide (CTAB). Add 170 ml to 75 ml of ethanol. After the bubbles disappear, add 0.1 ml of ammonia water and stir at 35°C and 500 rpm. 5 minutes, and then slowly add 0.2 ml tetraethyl orthosilicate (TEOS) dropwise. At this time, adjust the temperature of the water bath to 60°C. After reacting for 24 to 48 hours, centrifuge, and wash the sample three times with absolute ethanol to obtain inorganic Silicon oxide nanoparticles.

2. Synthesis of eccentric hollow mesoporous organic silicas (JMONs)

Step 1: Take 3.5 mg of the inorganic silica nanoparticles prepared in the previous steps, centrifuge at 10000 rpm, disperse in 0.5 ml ethanol, 8.5 ml deionized water, 0.015 g CTAB, 0.2~0.7 ml ammonia water mixture, and Stir evenly for 30 minutes at 35°C at 500 rpm, then slowly add 0.04ml of 1,2-bis(triethoxysilyl)ethane (BTSE) dropwise, continue stirring for 3 hours, centrifuge, and use ethanol to remove the product. Wash three times and once with deionized water;

Step 2: Dissolve the product obtained in the first step with 35 ml of deionized water in a 50 ml centrifuge tube, place it in a water bath at 80°C for 12 hours, then centrifuge, and wash twice with ethanol.

Step 3: Place the product obtained in the second step into a mixture of 200 ml ethanol and 0.4 ml concentrated hydrochloric acid, and react at 60°C and 1100 rpm. Repeat three times. The reaction times are 3 hours, 12 hours, and 3 hours respectively. The final product was washed three times with ethanol and dissolved in 30 ml of ethanol to obtain eccentric hollow mesoporous organic silica nanoparticles;

3. Modification of Ce6 and CTPP

First, 1 ml of carboxytriphenylphosphine CTPP (20 mg/ml), 1 ml of photosensitizer Ce6 (20 mg/ml) were mixed with 0.5 ml of bis[3-(triethoxysilyl)propyl]tetrasulfide EDC (20 mg/ml in N,N-dimethylformamide (DMF)) and 0.5 ml N-hydroxysuccinimide NHS (20 mg/ml in DMF) were mixed. The mixture was shaken on a shaker at room temperature for 3 hours to activate the carboxyl groups. Then 1 ml of nanoparticles was dispersed into the carboxyl-activated CTPP solution. After reacting for 12 hours, centrifuge and wash three times with water, then disperse into carboxyl-activated Ce6, react for 12 hours, and centrifuge and wash three times with water to obtain stable surface functionally modified JMONs-Ce6&CTPP.

4. Synthesis of self-carrying oxygen eccentric hollow mesoporous organic silica nanoparticles (JMONs- Ce6&CTPP@PFC)

The modified 1mg JMONs-Ce6&CTPP prepared in the previous steps was centrifuged at 10000 rpm, washed twice with deionized water, then quickly added 0.1 ml PFC solution after vacuuming, and ultrasonicated in an ice bath (below 4°C) for 2 minutes to obtain JMONs- Ce6&CTPP@PFC, dispersed in PBS for later use.

Figure 3a~d uses transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to observe the prepared MSNs nanoparticles. Nanoparticles have good dispersion and uniform size, with a particle size of approximately 200 nm.

Figure 4a~l shows the transmission electron microscope (TEM), scanning electron microscope (SEM), water and kinetic size, element distribution, Fourier transform infrared spectrum, and nitrogen adsorption and desorption curves of eccentric hollow mesoporous organic silica nanoparticles. image. It can be seen from the figure that the nanoparticles have good dispersion and uniform size, with a particle diameter of about 320 nm. The carbon, oxygen, and silicon elements in the structure are evenly distributed, and there are infrared characteristic peaks of organic components, and there is a uniform medium on the surface. The pore size is 2.3 nm.

Figure 5a~f shows the UV-visible absorption spectrum, hydration kinetic size, surface potential and element distribution images of eccentric hollow mesoporous organic silica nanoparticles modified with Ce6 and CTPP. The appearance of the Ce6 characteristic peak of JMONs-Ce6&CTPP material in the UV-visible absorption spectrum indicates the successful modification of Ce6 on the eccentric hollow organic silica. The hydration kinetic size and potential change images reflect the size changes and surface potential of the particles during each stage of the process. , the element distribution image shows that phosphorus elements are evenly distributed on the eccentric hollow organic silicon oxide.

Figure 6 shows the biocompatibility test of JMONs-Ce6&CTPP. Mouse breast cancer cells (4T1 cells) were used for the experiment. The concentration ranged from 0-200 μg/l. The cell activity reached more than 80% after incubation for 24 h or even 48 h, proving that nanometer The particles have good biocompatibility.

Figure 7a~b shows the extracellular and intracellular singlet oxygen production of nanoparticles in different control groups. The results show that after applying a 660 nm excitation light source, the JMONs-Ce6&CTPP nanoparticles loaded with PFC have more obvious extracellular and intracellular singlet oxygen. Oxygen production, the singlet oxygen production rate of the JMONs-Ce6&CTPP@PFC experimental group is 3.5 times or more than that of the control group.

Figure 8 shows the photodynamic effect of JMONs-Ce6&CTPP@PFC. As the concentration of different experimental groups increases, the cell activity of the four experimental groups gradually decreases. When the material concentration reaches 200 μg/ml, it is the same as loading PFC without adding an excitation light source. Or compared with the free Ce6 control group, JMONs-Ce6&CTPP@PFC has greater cancer cell killing efficiency, and the cell survival rate is 2/5 of the free Ce6 control group and less than 40%.

The photodynamic therapy nano-platform provided by the present invention has a large internal cavity and mesoporous channels, can efficiently load oxygen-carrying perfluorocarbon molecules and target them to mitochondria, and has high singlet oxygen production characteristics after specific wavelength laser irradiation. and cancer cell killing properties. The preparation method includes regulating its growth structure through a two-step silicon source method to obtain eccentric organic silica and a hydrothermal etching method to obtain eccentric mesoporous organic silica, and functionally modifying the mitochondrial targeting molecule triphenylphosphine (TPP) on its surface. ) and the photosensitizer chlorin e6 (Ce6), loading a low-boiling point tetradecafluorohexane liquid with self-carrying oxygen properties into the cavity. The eccentric hollow oxygen-carrying mesoporous organic silica nanoparticles combine the mitochondrial targeting function to improve the mitochondrial targeting ability, singlet oxygen production ability and photodynamic therapy effect. The preparation method provided by the invention is simple and easy to obtain, and the obtained product has a high yield, and has huge application potential in fields such as tumor photodynamic therapy.

The present invention has been disclosed above with preferred embodiments, but they are not intended to limit the present invention. Any technical solutions obtained by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the present invention.

Claims (8)

1. A self-carrying oxygen mitochondria-targeted photodynamic therapy nano-platform, characterized by including eccentric hollow mesoporous organic silica, mitochondrial targeting molecules and photosensitizers modified on the surface of eccentric hollow mesoporous organic silica, and loading Self-carrying oxygen perfluorocarbon molecules within the eccentric cavity of eccentric hollow mesoporous organosilica; The preparation method includes: using an inorganic silicon source to synthesize mesoporous inorganic silica with adjustable size, using an organic silicon source to coat the mesoporous inorganic silica, and then using a hydrothermal method to remove the inorganic silica to form an eccentric hollow mesoporous organic silica, by aminating the surface of the eccentric hollow mesoporous organic silica to jointly modify mitochondrial targeting molecules and photosensitizers, and by loading self-carrying oxygen perfluorocarbon molecules in the eccentric cavity of the eccentric hollow mesoporous organic silica , obtain a self-carrying oxygen mitochondria-targeted photodynamic therapy nanoplatform; Among them, the eccentric hollow mesoporous organic silica modified with mitochondrial targeting molecules and photosensitizers is mixed with self-carrying oxygen perfluorocarbon molecules under vacuum conditions, and then ultrasonicated in an ice bath to obtain self-carrying oxygen mitochondria-targeted photodynamic therapy. Nanoplatform. 2. A self-carrying oxygen mitochondria-targeted photodynamic therapy nano-platform according to claim 1, characterized in that the eccentric cavity size of the eccentric hollow mesoporous organic silica is 150-300 nm. 3. A self-oxygen-carrying mitochondria-targeted photodynamic therapy nano-platform according to claim 1, characterized in that the eccentric cavity surface of the eccentric hollow mesoporous organic silica has an ultra-thin mesoporous shell. 4. A self-carrying oxygen mitochondria-targeted photodynamic therapy nano-platform according to claim 1, characterized in that the eccentric hollow mesoporous organic silica has uniform mesoporous channels. 5. The self-carrying oxygen mitochondria-targeted photodynamic therapy nano-platform according to claim 1, characterized in that during the synthesis of eccentric hollow mesoporous organic silica, an organic silicon source is grown heterogeneously on the surface of mesoporous inorganic silica, Eccentric hollow mesoporous organic silica was obtained under the conditions of 35°C, 500rpm, and ammonia water and organic silicon source ratio of 1:100-25. 6. The self-carrying oxygen mitochondria-targeted photodynamic therapy nano-platform according to claim 1, characterized in that the inorganic silicon source is TEOS, the organic silicon source is BTSE, and the volume ratio of the two is 1:0.05- 0.5. 7. The self-carrying oxygen mitochondria-targeted photodynamic therapy nano-platform according to claim 1, characterized in that during the synthesis of eccentric hollow mesoporous organic silica, inorganic oxidation is removed by hydrothermal method in a water bath at 80°C for 12 hours. silicon to form eccentric hollow mesoporous organic silica. 8. Application of the self-carrying oxygen mitochondria-targeted photodynamic therapy nano-platform according to any one of claims 1 to 7 in the preparation of anti-tumor drugs.