CN113101365A

CN113101365A - Photodynamic nano platform with mitochondrion targeting characteristic and preparation method and application thereof

Info

Publication number: CN113101365A
Application number: CN202110335085.9A
Authority: CN
Inventors: 滕兆刚; 史文慧
Original assignee: Nanjing University of Posts and Telecommunications
Current assignee: Nanjing University of Posts and Telecommunications
Priority date: 2021-03-29
Filing date: 2021-03-29
Publication date: 2021-07-13

Abstract

The invention belongs to the technical field of photodynamic therapy, and discloses a photodynamic nano-platform with mitochondrial targeting properties and a preparation method and application thereof. The photodynamic nanoplatform with mitochondrial targeting properties is composed of flexible mesoporous organosilica, mitochondrial targeting molecules and photosensitizers. It has a shrunken surface, large internal cavity, and mesoporous pores, which can efficiently load drugs and Has high cellular uptake properties. The preparation method includes obtaining a flexible mesoporous organic silica shell layer by alkaline solution etching, and performing functional modification on the surface of mitochondrial targeting molecule and photosensitizer Ce6. Flexible mesoporous organosilicon nanoparticles combine mitochondria-targeting functions to enhance intracellular phagocytosis efficiency, mitochondria-targeting ability, and photodynamic therapy efficacy. The preparation method provided by the invention is simple and easy to obtain, and the obtained product has a high yield, and has great application potential in the fields of tumor photodynamic therapy and the like.

Description

Photodynamic nano platform with mitochondrion targeting characteristic and preparation method and application thereof

Technical Field

The invention belongs to the technical field of photodynamic therapy, and particularly relates to a photodynamic nano platform with a mitochondrion targeting characteristic, and a preparation method and application thereof.

Background

Photodynamic therapy is an emerging tumor treatment following surgical resection, chemotherapy, and radiation therapy. Photodynamic therapy medicines, namely photosensitizers can selectively act on tumor parts, have the advantages of small wound, good repeatability, no accumulated toxicity, less adverse reaction and the like, and show more and more obvious advantages in tumor treatment. Despite the broad prospects for tumor therapy, the clinical application of conventional photodynamic therapy still has many disadvantages. Firstly, most of the photosensitizers are aromatic molecules which are poorly soluble in water and tend to aggregate, thus easily causing skin phototoxicity. On the other hand, the singlet oxygen generated by the photosensitizer has small action range and short service life, and can act when not reaching organelles, thereby greatly restricting the killing effect of the photosensitizer. To solve these problems, nano-support platforms are increasingly used.

In recent years, flexible mesoporous organosilica nanomaterials have received extensive attention from biomedicine due to their material properties and special mechanical characteristics. As a nano-carrier platform, the mesoporous organic silicon oxide nano-particles have the characteristics of adjustable mesoporous structure, large specific surface area, good dispersibility, uniform size, biodegradability, high biocompatibility and the like, and become ideal carriers of various molecular imaging probes and anticancer therapeutic drugs. In the design of nanoparticles, mechanical properties play an important role. The flexibility is taken as an important mechanical property of the nano particles, and plays an important role in various biological processes such as blood circulation, tumor tissue infiltration, cell internalization and metabolism. However, the flexible organic silicon oxide nanoparticles have single function as a nano-drug carrier, so that the tumor treatment effect is difficult to achieve, and the characteristics of mitochondria targeting and photodynamic therapy are combined, so that the flexible mesoporous organic silicon oxide nanoparticles are widely researched in biomedical application, but more new applications are still to be explored.

Disclosure of Invention

The invention aims to provide a preparation method of a flexible mesoporous organic silicon oxide nano delivery platform loaded with carboxyl triphenyl phosphorus (CTPP) and a photosensitizer ce 6. The nano platform enhances the intratumoral uptake and simultaneously improves the loading efficiency of Ce6, and in addition, the mitochondrial targeting effect of triphenylphosphine further improves the killing efficiency of Ce6 cells. Based on the basic characteristics of flexible mesoporous silica and photodynamic therapy, the invention provides a preparation method of a flexible mesoporous organic silica nano delivery platform and a method for loading triphenylphosphine and a photosensitizer ce 6. The accumulation in the tumor is improved, and the mitochondria are targeted, so that the photodynamic treatment effect is further enhanced.

The invention is realized by the following technical scheme:

a photodynamic nano platform with a mitochondrion targeting characteristic comprises flexible mesoporous organic silicon oxide, mitochondrion targeting molecules and a photosensitizer.

Further, the mitochondrion targeting molecule is triphenylphosphine.

Further, the photosensitizer is Ce 6.

Furthermore, the flexible mesoporous organic silicon oxide shell is doped with disulfide bonds.

Furthermore, the thickness of the flexible mesoporous organic silicon oxide shell layer is 20-30 nm.

A preparation method of a photodynamic nano platform with a mitochondrion targeting characteristic comprises the steps of synthesizing mesoporous organic silicon oxide containing a disulfide bond by using an organic silicon source, breaking the disulfide bond to connect amino to obtain aminated nano particles, modifying triphenyl phosphorus and a photosensitizer Ce6 on the surfaces of the aminated nano particles together to obtain mesoporous organic silicon oxide nano particles with CTPP and Ce6, and etching the mesoporous organic silicon oxide nano particles in an alkaline solution to form high tumor uptake to obtain the photodynamic flexible nano platform with the mitochondrion targeting function.

Further, in the process of synthesizing the mesoporous organic silicon oxide containing disulfide bonds, an organic silicon source with disulfide bonds is doped in the flexible mesoporous organic silicon oxide shell, the disulfide bonds are broken under the conditions of 40 ℃ and 800rpm and N2 full filling, and the amino groups of the maleamide are connected to obtain the aminated nanoparticles.

Further, after the mesoporous organic silicon oxide nanoparticles modified with CTPP and Ce6 are centrifuged, adding alkali liquor, placing the mixture in a shaking table for 10-20 min, and washing the mixture for 2-4 times to obtain flexible mesoporous organic nanoparticles, wherein the flexible mesoporous organic nanoparticles are dispersed in ethanol.

Furthermore, the organic silicon source is TETS and TEOS, and the volume ratio of TETS to TEOS is 1: 2-3.

The invention also provides application of the photodynamic nano platform with the mitochondrion targeting characteristic in preparing antitumor drugs.

A preparation method of a photodynamic nano platform with a mitochondrion targeting characteristic comprises the following steps:

(1) synthesis of organosilica nanoparticles (MONs): adding 0.16g of hexadecyl trimethyl ammonium bromide (CTAB) into 30ml of ethanol and 75ml of deionized water, adding 1ml of ammonia water, reacting for 1 hour at 35 ℃ and at the rotating speed of 1100rpm, adding 0.1ml of 1, 3-ethoxy silicon-based tetrasulfide (TETS) and 0.25ml of tetraethyl orthosilicate (TEOS) which are mixed with a silicon source, reacting for 24 hours, and centrifuging and washing for 3 times to obtain organic silicon oxide nanoparticles;

(2) surface-modified amino group: first, the disulfide bond was cleaved by adding 2.4ml of water, 8.8ml of dioxane, 0.4g of triphenylphosphine and 160. mu.l of concentrated hydrochloric acid to a sample, and reacting for 2 hours at 40 ℃ and 800rpm filled with N2. After 4mg of maleimido amino group was added to the disulfide-bond-cleaved sample, the reaction was carried out for 12 hours, and centrifuged and washed 3 times.

(3) Activating carboxyl and connecting: first, 1ml of carboxytriphenylphosphine CTPP (20mg/ml), 1ml of the photosensitizer Ce6(20mg/ml) were mixed with 0.5ml of bis [3- (triethoxysilyl) propyl ] tetrasulfide EDC (20mg/ml in N, N-Dimethylformamide (DMF)) and 0.5ml of N-hydroxysuccinimide NHS (20mg/ml in DMF), respectively. The mixture was shaken at room temperature for 3 hours to activate the carboxyl group. Then 1ml of nanoparticles were dispersed into the carboxy-activated CTPP solution. After 12h of reaction, centrifugal washing is carried out for 3 times, and the mixture is dispersed into Ce6 activated by carboxyl, and the stable MONs-CTPP-Ce6 modified by surface function is obtained after 12h of reaction and 3 times of centrifugal washing.

(4) Synthetic flexible organosilica nanoparticles (SMONs-CTPP-Ce 6): centrifuging 2mg MONs-CTPP-Ce6 with modified surface at 10000rpm, dispersing in 1ml water, adding 100 μ L1M NaOH solution, placing in a shaking table for 15min, washing with water for 3 times to obtain flexible mesoporous organic nanoparticles (SMONs-CTPP-Ce6), and dispersing in ethanol.

Advantageous effects

1. The mesoporous organic silicon oxide has rich mesopores, and the photodynamic nano platform provided by the invention has a shrunken surface, a larger internal cavity and a mesoporous pore channel, can be used for loading the drug with high efficiency, has the characteristic of high-efficiency cell uptake, and is beneficial to loading and transporting the drug;

2. the flexible mesoporous organic silicon oxide nano-particles are prepared by an alkaline solution etching method, the requirements on equipment in the process are low, the cost is low, and the environment is friendly; the preparation method provided by the invention 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;

3. the flexible material has more excellent in vivo circulation and tumor uptake capacity;

4. after the surface modification is finished, the mitochondrion targeting obviously improves the curative effect of photodynamic therapy; the flexible mesoporous organic silicon nano-particles combine the mitochondrion targeting function, and improve the endocytosis efficiency, the mitochondrion targeting capability and the photodynamic treatment effect of cells.

Drawings

FIG. 1 is a structural formula of a photosensitizer Ce 6;

FIG. 2 is a structural formula of Carboxytriphenylphosphonium (CTPP);

fig. 3 is a working principle of the preparation and synthesis of the mesoporous organic silicon oxide nano platform and the preparation method of the supported photosensitizer Ce6 and Carboxyl Triphenylphosphine (CTPP) in embodiment 1 of the present invention;

FIG. 4 is a Scanning Electron Microscope (SEM) and a Transmission Electron Microscope (TEM) representation of the prepared flexible mesoporous silica nanoparticles in example 1 of the present invention;

FIG. 5 is a high-power transmission electron microscope bright field and element distribution image of the prepared flexible mesoporous silica nanoparticles in example 1 of the present invention;

FIG. 6 is a potential diagram and an ultraviolet absorption spectrum diagram of the flexible mesoporous silica nanoparticles prepared in example 1 according to the present invention at different loading stages;

FIG. 7 is a cytocompatibility chart of the surface-modified flexible mesoporous silica nanoparticles prepared in example 1 of the present invention;

fig. 8 is a comparative graph of targeted photodynamic therapy of the prepared surface-modified flexible mesoporous silica nanoparticles in example 1 of the present invention.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following further describes the technical solution of the present invention with reference to the drawings and the embodiments.

With reference to fig. 1, 2 and 3, a preparation method of a flexible mesoporous organosilica nano-delivery platform loaded with CTPP and a photosensitizer ce6 is specifically described.

Example 1: synthesis of Mesoporous Organosilica Nanoparticles (MONs):

all synthetic experiments are completed in a water bath kettle; first, 75ml of deionized water, 30ml of ethanol, 1ml of concentrated ammonia water and 0.16g of cetyltrimethylammonium bromide were sequentially added to a reaction flask, stirred at 1100rpm for 1 hour at 35 ℃, and then a mixed solution of 0.25ml of TEOS and 0.1ml of TETS was added to the above reaction flask, and stirring was continued for 24 hours to obtain a white suspension, which was separated and washed 3 times with ethanol. Dispersing the obtained nano particles in a mixed solution of 120ml of ethanol and 240 mul of concentrated hydrochloric acid, and heating for 12h at the temperature of 60 ℃ to remove the surfactant, thereby finally obtaining the mesoporous organic silicon oxide nano particles.

Example 2

Surface modification: it is necessary to modify the surface of the particles with amino groups and then to react with the carboxyl groups CTPP and Ce 6.

First, disulfide bond cleavage: washing 10 mg of nano material with deionized water, dispersing in a mixed solution of 1.2ml of deionized water and 4.4ml of dioxane, adding 0.19 g of triphenylphosphine and 80 μ l of concentrated hydrochloric acid, ultrasonically mixing for 10min, placing in a water bath, reacting for two hours at 40 ℃ and 800rpm in a nitrogen environment, washing once with alcohol, and washing twice with water.

In the second step, the treated sample was dispersed in 1.2ml of water, 0.12ml of DMF and 4mg of NH2-MAL, respectively, reacted for 12 hours, washed three times with deionized water, and then dispersed in 1ml of water.

In the third step, CTPP was activated and attached to the carboxyl group of ce 6. First, 1ml of CTPP (20mg/ml), 1ml of ce6(20mg/ml) were mixed with 0.5ml of EDC (20mg/ml in DMF) and 0.5ml of NHS (20mg/ml in DMF), respectively. The mixture was shaken at room temperature for 3 hours to activate the carboxyl group. Then 1ml of nanoparticles were dispersed into the carboxy-activated CTPP solution. After 12h of reaction, centrifugal washing is carried out for 3 times, and the mixture is dispersed into Ce6 activated by carboxyl, and the stable MONs-CTPP-Ce6 modified by surface function is obtained after 12h of reaction and 3 times of centrifugal washing.

Example 3

Preparation of Flexible MONs-CTPP-Ce6

Centrifuging 2mg MONs-CTPP-Ce6 with modified surface at 10000rpm, dispersing in 1ml water, adding 100 μ L of 1M NaOH solution, placing in a shaking table for 15min, washing with water for 3 times to obtain flexible mesoporous organic nanoparticles (SMONs-CTPP-Ce6), and dispersing in ethanol.

FIGS. 4 a-c, the resulting SMONs-CTPP-Ce6 nanoparticles were observed using Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). The flexible nanoparticles had good dispersibility, were uniform in size, had a particle size of about 200nm, and significant surface shrinkage was observed, with a silicon layer thickness of about 25 nm.

Fig. 5 d-f are high-power transmission electron microscope bright field and element distribution images of the flexible mesoporous organosilicon nanoparticles, and the element distribution images show that phosphorus is uniformly distributed on the surfaces of the flexible mesoporous organosilicon nanoparticles.

Fig. 6a shows the potential change in different loading processes, the surface modified amino group, CTPP and Ce6 have obvious positive-negative conversion, and the potential does not change after the particle etching, indicating that the modified molecules are stably connected.

FIG. 6b shows UV testing of free Ce6 and SMONs-CTPP-Ce6, since free Ce6 shows a distinct absorption peak at 660nm, while SMONs-CTPP-Ce6 shows the same peak, indicating successful loading of photosensitizer Ce 6.

FIG. 7 shows that SMONs-CTPP-Ce6 is used as a biocompatibility test, mouse breast cancer cells (4T1 cells) are selected for experiments, the concentration is 0-1000 mug/l, the cell activity reaches more than 80%, and the nanoparticles are proved to have good biocompatibility.

Fig. 8 is a graph showing the photodynamic effect of SMONs-CTPP-Ce6, and the cell activities of the three experimental groups gradually decreased with the increase of the concentration of the photosensitizer Ce6, and when the concentration of Ce6 reached 20 μm/ml, the SMONs-CTPP-Ce6 had a greater killing efficiency and the cell survival rate was 1/2 and less than 20% of that of the control group of free Ce6, compared with the control group of no mitochondrial targeting and free Ce 6.

The above experiments describe the basic principles, main features and advantages of the present invention. However, the above description is only an example of the present invention, the technical features of the present invention are not limited thereto, and any other embodiments that can be obtained by those skilled in the art without departing from the technical solution of the present invention should be covered by the claims of the present invention.

Claims

1. A photodynamic nanoplatform with mitochondrial targeting characteristics, characterized in that, the photodynamic nanoplatform comprises flexible mesoporous organosilica, mitochondrial targeting molecule and photosensitizer.

2 . The photodynamic nano-platform with mitochondrial targeting properties according to claim 1 , wherein the mitochondrial targeting molecule is triphenylphosphine. 3 .

3. The photodynamic nanoplatform with mitochondrial targeting properties according to claim 1, wherein the photosensitizer is Ce6.

4 . The photodynamic nanoplatform with mitochondrial targeting properties according to claim 1 , wherein the flexible mesoporous organosilica shell is doped with disulfide bonds. 5 .

5 . The photodynamic nanoplatform with mitochondrial targeting properties according to claim 1 , wherein the thickness of the flexible mesoporous organosilica shell layer is 20-30 nm. 6 .

6. The method for preparing a photodynamic nanoplatform with mitochondrial targeting properties according to any one of claims 1 to 5, characterized in that, using an organosilicon source to synthesize mesoporous organosilicon containing disulfide bonds, disconnecting Aminated nanoparticles were obtained by connecting amino groups with disulfide bonds, and triphenylphosphorus and photosensitizer Ce6 were co-modified on the surface of the aminated nanoparticles to obtain mesoporous organosilica nanoparticles modified with CTPP and Ce6. After etched, high tumor uptake was formed, and a photodynamic flexible nanoplatform with mitochondrial targeting function was obtained.

7. The method for preparing a photodynamic nanoplatform with mitochondrial targeting characteristics according to claim 6, wherein in the process of synthesizing the mesoporous organosilicon containing disulfide bonds, in the flexible mesoporous organosilicon shell Doping an organosilicon source with a disulfide bond, breaking the disulfide bond at 40 °C, 800 rpm, filled with ^N2 , and connecting the maleamido amino group to obtain aminated nanoparticles.

8. the preparation method of the photodynamic nano-platform with mitochondrial targeting characteristic according to claim 6, is characterized in that, after the mesoporous organosilica nanoparticle of modified CTPP and Ce6 is centrifuged, adds lye and is placed in shaking table 10 . ~20min, washed 2~4 times with water to obtain flexible mesoporous organic nanoparticles, which were dispersed in ethanol.

9 . The method for preparing a photodynamic nanoplatform with mitochondrial targeting properties according to claim 6 , wherein the organosilicon source is TETS and TEOS, and the volume ratio of TETS and TEOS is 1:2-3. 10 .

10 . The application of the photodynamic nanoplatform with mitochondrial targeting properties according to any one of claims 1 to 5 in the preparation of antitumor drugs. 11 .