CN112694577B - Imprinted mesoporous material and preparation method and application thereof - Google Patents

Abstract

The invention discloses a imprinted mesoporous material, which is mesoporous SiO with the outer diameter of 100-200 nm and subjected to C ═ C surface modification and sialic acid surface imprinting treatment ₂ And (3) granules. The preparation method comprises the following steps: firstly preparing mesoporous SiO ₂ Then using mesoporous SiO modified by surface double bond ₂ The nanospheres are used as carriers, sialic acid to be detected is used as template molecules to prepare mesoporous silicon-based molecularly imprinted microspheres, and then the template molecules are removed through dialysis under acidic conditions to prepare the molecularly imprinted microspheres. Then embedding the tumor therapeutic drug adriamycin into the microsphere pore canal by a solvent permeation method for tumor targeted therapy. The imprinted mesoporous material can directly reach the focus by loading the drug, not only has the effect of killing cancer cells, but also has the effect of causing cancer cell apoptosis, and realizes the aim of targeted therapy on tumors.

Description

A kind of imprinted mesoporous material and its preparation method and application

technical field

The invention belongs to an imprinted mesoporous material, a preparation method and application thereof, and belongs to the technical field of tumor cell targeted drug development and preparation.

Background technique

The liver is one of the predilection sites for tumors, benign tumors are rare, and metastatic tumors are more common in malignant tumors. Primary tumors can occur in hepatocyte cords, bile duct epithelium, blood vessels or other mesodermal tissues. Most metastatic tumors are metastatic carcinomas, and a few are metastatic sarcomas. Since the liver is the largest solid organ in the human body, it undertakes various important metabolic functions of the human body. Therefore, once the liver is malignant, it will lead to serious consequences that endanger life. Also, because the liver has abundant blood supply, it is closely related to the important blood vessels of the human body, and the onset of malignant tumors in the liver is hidden and grows rapidly, so the treatment is very difficult, and the overall curative effect and prognosis are not very satisfactory.

Doxorubicin (DOX) is one of the most commonly used anticancer drugs, especially for metastatic cancer cells. However, gastrointestinal reactions, cardiotoxicity, and other non-specific drug side effects due to cytotoxicity limit the efficacy of clinical treatment. Combining chemotherapeutic drugs with nanocarriers can control the release of therapeutic agents during blood transport, allowing them to reach the cancer tissue before being released. Therefore, adverse reactions can be reduced while enhancing the anticancer effect. Due to the high specific surface area and ordered mesoporous structure of mesoporous _SiO2 particles, they enable efficient drug delivery.

Molecular imprinting technology is a technology for preparing high molecular polymer materials with specific affinity and recognition ability for target molecules. Molecularly imprinted polymers prepared by traditional methods have disadvantages such as difficult extraction of target molecules, low adsorption capacity and poor kinetic properties due to their highly cross-linked structure. Imprinting target molecules on the surface of carrier materials can greatly improve the adsorption capacity and kinetic properties of molecularly imprinted polymers, and can be used for targeted anticancer drugs, eye peptide drugs, oral insulin and enantioselective racemate drugs. controlled release. Mesoporous silica has the advantages of large specific surface area/easy modification, good stability and high mechanical strength, and is very suitable as a carrier material for surface molecularly imprinted polymers. However, in the prior art, there is no mild, biocompatible and widely applicable imprinted mesoporous material.

SUMMARY OF THE INVENTION

One of the objects of the present invention is to provide a traced mesoporous material. The specific technical solutions are as follows:

An imprinted mesoporous material, the imprinted mesoporous material is a mesoporous SiO ₂ particle that has undergone C=C surface modification and sialic acid surface imprinting treatment, and the particle has an outer diameter of 100-200 nm.

The second object of the present invention is to provide a preparation method of the imprinted mesoporous material. The specific technical solutions are as follows:

The preparation method of the imprinted mesoporous material comprises the following steps:

( ₁ ) Preparation of mesoporous SiO

The porogen cetyltrimethylammonium bromide (CTAB) and ammonium fluoride (NHF ₄ ) are dispersed in deionized water, slowly add tetraethyl orthosilicate (TEOS), and react at 70～100℃ for 1～3h , then repeatedly washed with deionized water and ethanol, centrifuged and dried to obtain complete SiO ₂ particles, disperse the SiO ₂ particles in ethanol, add hydrochloric acid, reflux at 70 ~ 100 ° C for 18 ~ 36h, and then use Repeated washing and centrifugation with ionized water and ethanol to remove the eluted porogen to obtain mesoporous SiO ₂ particles;

( ₂ ) Silanation modification of mesoporous SiO2 (MSNs)

The mesoporous _SiO2 particles were uniformly dispersed in ethanol to form an ethanolic solution of mesoporous _SiO2 particles, which was mixed with 3-(methacryloyloxy)propyltrimethoxysilane (MPTS) and ethanol at 40-60 °C The reaction is carried out for 8-18 hours, and then centrifuged and washed repeatedly with deionized water and ethanol to remove unreacted components, and after drying, C=C surface-modified mesoporous SiO ₂ particles are obtained;

(3) Preparation of sialic acid surface-imprinted mesoporous SiO particles (MIP _- MSNs)

The C=C surface _- modified mesoporous SiO particles obtained in the previous step, as well as phosphate buffer solution (PBS), sialic acid (SA), 3-methacrylamidophenylboronic acid (MAPBA), N -Isopropylacrylamide (NIPAM), Acrylamide (AAM), N-(3-aminopropyl)methacrylamide hydrochloride, N,N'-methylenebisacrylamide (BIS), ultrasonic dispersion After homogenization, magnetic stirring at room temperature, and then adding ammonium persulfate solution (APS) and N,N,N',N'-tetramethylethylenediamine solution ( TMEDA), place the reaction vessel in a water bath at 35 to 40 °C for 12 to 36 h, collect and dialyze by centrifugation, and collect the particles by centrifugation again to obtain mesoporous SiO ₂ particles (MIP-MSNs) imprinted on the surface of sialic acid;

In step (1), the ratio of hexadecyl trimethyl ammonium bromide, ammonium fluoride, ethyl orthosilicate, ethanol, hydrochloric acid is: (400-500mg): (700-800mg): (2-3ml) :(90-110mL):(1.5-3mL);

Step (2) The ratio of mesoporous SiO ₂ particles, the sum of ethanol, and 3-(methacryloyloxy)propyltrimethoxysilane is: (450-500mg):(50-70mL):(1-3mL);

C=C surface-modified mesoporous SiO particles in step ( ₃ ), phosphate buffer solution, sialic acid, 3-methacrylamidophenylboronic acid, N-isopropylacrylamide, acrylamide, N- The ratio of (3-aminopropyl)methacrylamide hydrochloride, N,N'-methylenebisacrylamide, ammonium persulfate, N,N,N',N'-tetramethylethylenediamine is :(450-500mg):(25-35mL):(15-20mg):(10-13mg):(80-85mg):(23-27mg):(10-12mg):(6-10mg):( 45-55 μL): (95-105 μL).

Preferably, the time of the magnetic stirring in step (3) is 2-6h.

Preferably, in step (3), the time for argon ventilation is 20-60 min.

The third object of the present invention is to provide the application of the imprinted mesoporous material. The specific technical solutions are as follows:

The imprinted mesoporous material is applied to tumor cell targeted drug delivery.

Preferably, the tumor cell targeting drug is doxorubicin.

More preferably, the method of application comprises the steps of: dissolving the sialic acid surface-imprinted mesoporous SiO ₂ particles and doxorubicin in a phosphate buffer solution by a solvent permeation method, magnetically stirring at room temperature to make the mixing complete, and centrifuging. The supernatant was discarded, and the particles were collected to prepare doxorubicin-loaded nanoparticles for targeted therapy of tumor cells; the sialic acid surface-imprinted mesoporous SiO ₂ particles, doxorubicin, and phosphate buffer solution The ratio is: (15-25 mg): (3-7 mg): (8-12 mL).

More preferably, the time of the magnetic stirring is 5-20h.

The beneficial effects of the present invention

The present invention provides a molecularly imprinted material, sialic acid surface-imprinted mesoporous SiO ₂ particles (MIP-MSNs). A molecularly imprinted polymer layer is grafted on the surface of the C=C-modified mesoporous SiO ₂ particles. The imprinting process is performed on the surface of mesoporous silica, and the synthesized imprinted material also has a larger specific surface area, a faster mass transfer rate, more surface imprinted active sites are exposed, and the selectivity is improved. The surface imprinting layer blocks doxorubicin in the mesopores and releases it when combined with tumor cells. Therefore, the imprinted mesoporous material-loaded drug (such as doxorubicin) of the present invention can directly reach the lesions, and not only has the ability to kill cancer cells. It also has the effect of causing apoptosis of cancer cells, so as to achieve the purpose of targeted therapy of tumors.

Description of drawings

Fig. 1 is the scanning electron microscope picture of mesoporous silica in Example 1 of the present invention;

2 is a TEM image of mesoporous silica in Example 1 of the present invention;

Fig. 3 is the infrared spectrogram of the mesoporous silica of the embodiment of the present invention 1;

Fig. 4 is the maximum drug loading figure under different doxorubicin concentrations in Example 2 of the present invention.

Detailed ways

Example 1

( ₁ ) Preparation of mesoporous SiO

Accurately weigh 450 mg of cetyltrimethylammonium bromide (CTAB) and 750 mg of ammonium fluoride (NHF ₄ ) and disperse them in 123 mL of deionized water. After the ultrasonic dispersion is uniform, slowly add 2.25 mL of ethyl orthosilicate (TEOS) , mixed evenly, heated to 80°C, and reacted for 2h. After the reaction is completed, repeatedly wash with deionized water and ethanol to remove unreacted components and other impurities, and obtain complete _SiO2 particles after centrifugation and drying. The dried SiO ₂ particles were dispersed in 100 mL of ethanol, 2 mL of hydrochloric acid was added, and refluxed at 90 °C for 24 h to remove the porogen cetyltrimethylammonium bromide. After the reaction, wash and centrifuge repeatedly with deionized water and ethanol to remove the eluted porogen, and obtain _SiO2 particles with hollow mesoporous structure.

The nanoparticles were examined by scanning electron microscope and transmission electron microscope. The results of electron microscopy showed that the mesoporous silica particles were uniform, with the same pore size and uniform distribution (as shown in Figures 1 and 2).

( ₂ ) Silanation modification of mesoporous SiO2 (MSNs)

Weigh 500 mg of mesoporous SiO particles and evenly disperse them in 10 mL of ethanol, add ₂ mL of 3-(methacryloyloxy)propyltrimethoxysilane (MPTS), 50 mL of ethanol, ethanol solution of mesoporous SiO particles to a single _- necked flask, 50 The reaction was carried out at ℃ for 12h. After the reaction was completed, centrifugation and washing with deionized water and ethanol were repeated to remove unreacted components, and C=C surface-modified mesoporous SiO ₂ particles were obtained after drying.

(3) Sialic acid surface-imprinted mesoporous SiO particles (MIP _- MSNs)

To a single-necked flask, add 500 mg of C=C surface _- modified mesoporous SiO particles, 34 mL of phosphate buffered solution (PBS), 18.6 mg of sialic acid (SA), 11.4 mg of 3-methacrylamidobenzene boronic acid (MAPBA), 81mg of N-isopropylacrylamide (NIPAM), 25.4mg of acrylamide (AAM), 10.6mg of N-(3-aminopropyl)methacrylamide hydrochloride, 7.8mg The N,N'-methylenebisacrylamide (BIS) was uniformly dispersed by ultrasonic, and then magnetically stirred at room temperature for 4 h to make the template and functional monomer self-assemble. Then, 51 μL of ammonium persulfate solution (APS) and 102 μL of N,N,N',N'-tetramethylethylenediamine solution ( TMEDA). The flask was placed in a water bath at 37°C for 24h. After the reaction was completed, the samples were collected by centrifugation, and dialyzed against ice water with a 300,000 MW dialysis bag to remove template molecules. After the template molecules were eluted, they were collected by centrifugation and freeze-dried to obtain sialic acid surface-imprinted mesoporous SiO ₂ particles (MIP-MSNs).

As a control, non-imprinted mesoporous SiO ₂ particles (NIP-MSNs) were prepared in the same method without adding template sialic acid molecules, and the monomer components and synthesis steps used were the same as above.

(4) Loading chemotherapeutic drug doxorubicin (MIP-MSNs@DOX)

20 mg of sialic acid surface-imprinted mesoporous SiO ₂ particles (MIP-MSNs) and 5 mg of doxorubicin were dissolved in 10 mL of phosphate buffer solution by solvent permeation method, magnetically stirred at room temperature for 12 h, mixed completely, and discarded by centrifugation The supernatant was collected, and the particles were collected to prepare doxorubicin-loaded nanoparticles.

The Fourier transform infrared spectrum shows that the surface of the mesoporous silica particles has been successfully modified with C=C, and the surface molecularly imprinted layer has been successfully grafted (as shown in Figure 3).

Example 2

The optimal loading efficiency of doxorubicin on the same weight of sialic acid surface-imprinted mesoporous _SiO2 particles (MIP-MSNs) nanomaterials under different doxorubicin concentrations was confirmed. The specific operation is briefly described as follows: 1 mg of MIP-MSNs were uniformly dispersed in doxorubicin solutions of different concentrations, shaken and mixed at 37 °C for 24 h, centrifuged at 12,000 rpm for 5 min, and the supernatant was taken for spectrophotometer determination of MIP-MSNs in Loading efficiency in doxorubicin solutions of different concentrations. Figure 4 shows that at 2.5 mg/mL, the MIP-MSNs had the largest drug loading and the centrifuged supernatant had the lowest drug content (Figure 4).

Example 3

In vitro uptake experiment of hepatoma cells (HepG-2) to specific targeting drug-loaded MSNs system. Doxorubicin is the most commonly used tumor chemotherapy drug. It has good water solubility and can emit red fluorescence under ultraviolet irradiation. Therefore, the situation of nanoparticles in contact with cells can be judged by observing the fluorescence intensity with a fluorescence microscope.

When HepG-2 cells were passaged for 24 hours and the cell adhesion rate reached about 80%, MIP-MSNs and NIP-MSNs loaded with doxorubicin were added respectively, and after culturing at 37°C for 24 hours, the nuclei were stained with Hoechst 33342 dye, and the nuclei were stained with a fluorescence microscope. The amount of doxorubicin entering the cells was observed for the two mesoporous silica nanomaterials.

Hoechst 33342 is a blue dye that stains cell nuclei. Doxorubicin has the characteristics of autofluorescence and emits red fluorescence under ultraviolet light. The cultured hepatoma cells HepG-2 were incubated with drug-loaded MIP-MSNs and NIP-MSNs at 37°C for 24 h, and the amount of adriamycin entering the cells was observed by fluorescence microscope (red fluorescence intensity). The experimental results showed that the amount of MIP-MSNs entering cells was significantly more than that of NIP-MSNs.

Example 4

Effects of specific targeting drug-loaded MSNs system in inhibiting proliferation and promoting apoptosis of liver cancer cells in vitro. Different concentrations of free doxorubicin, mesoporous silica-loaded doxorubicin (MSNs-DOX) and mesoporous silica-loaded doxorubicin on surface modified imprinted layer (MIP-DOX) were detected by CCK-8 and apoptosis assays After MSNs and NIP-MSNs) acted on tumor cells (HepG-2) for 48h, the cell survival rate and apoptosis rate were observed, and the therapeutic effect of mesoporous silica system was observed.

The results showed that the free doxorubicin had the strongest toxicity to the cells, because the free doxorubicin would directly enter the cells and directly cause the cells to die, and also cause the apoptosis of normal cells. After the mesoporous silica drug-loading system is loaded with doxorubicin into cells, due to the encapsulation and specific recognition of the surface imprinting layer, the nanoparticles will only enter the tumor cells and slowly release doxorubicin. The results confirmed that the mesoporous silica drug-loading system (MIP-MSNs@DOX) with surface-modified imprinted layer had stronger killing effect on tumor cells than the unmodified mesoporous silica drug-loading system (NIP-MSNs@DOX).

The above experimental results show that the tumor cell-targeted sialic acid-imprinted mesoporous silica drug-loading system has excellent in vitro anti-hepatoma therapeutic effect and good biocompatibility. Furthermore, this system did not show significant cytotoxicity to other normal cells. Further, the imprinted layer on the surface of mesoporous silica can increase the drug uptake of liver cancer cells and achieve high targeted therapy.

Claims (4)

1. The imprinted mesoporous material is characterized by being mesoporous SiO subjected to C ═ C surface modification and sialic acid surface imprinting treatment ₂ Particles, the outer diameter of the particles being 100 to 200 nm; the preparation method comprises the following steps of,

(1) mesoporous SiO ₂ Preparation of

Dispersing pore-foaming agents Cetyl Trimethyl Ammonium Bromide (CTAB) and ammonium fluoride in deionized water, slowly adding Tetraethoxysilane (TEOS), reacting for 1-3 h at 70-100 ℃, then repeatedly washing with the deionized water and ethanol, centrifuging and drying to obtain complete SiO ₂ Particles of said SiO ₂ Dispersing the particles in ethanol, adding hydrochloric acid, refluxing for 18-36 h at 70-100 ℃, repeatedly washing and centrifuging by using deionized water and ethanol, and removing the pore-forming agent eluted to prepare the mesoporous SiO ₂ A particle;

(2) mesoporous SiO ₂ Modified by silanization (MSNs)

Making mesoporous SiO ₂ The particles are uniformly dispersed in ethanol to form mesoporous SiO ₂ Reacting the ethanol solution of the particles with 3- (methacryloyloxy) propyltrimethoxysilane (MPTS) and ethanol for 8-18 h at 40-60 ℃, then repeatedly centrifuging and washing with deionized water and ethanol to remove unreacted components, and drying to obtain the C ═ C surface modified mesoporous SiO ₂ Particles;

(3) preparation of sialic acid surface imprinted mesoporous SiO ₂ Particle (MIP-MSNs)

Adding the C ═ C surface modified mesoporous SiO obtained in the previous step into a reaction vessel ₂ Particles, Phosphate Buffer Solution (PBS), Sialic Acid (SA), 3-methylacrylamidophenylboronic acid (MAPBA), N-isopropylacrylamide (NIPAM), acrylamide (AAM), N- (3-aminopropyl) methacrylamide hydrochloride and N, N' -methylenebisacrylamide (BIS) were ultrasonically dispersed uniformly, and then stirred magnetically at room temperature, and argon gas was introduced into the reaction system while adding the mixture to the reaction systemAmmonium Persulfate Solution (APS) and N, N, N ', N' -tetramethylethylenediamine solution (TMEDA), placing the reaction vessel in 35-40 ℃ water bath for reaction for 12-36 h, centrifugally collecting and dialyzing, centrifugally collecting particles again to obtain the sialic acid surface imprinted mesoporous SiO ₂ Particles (MIP-MSNs);

in the step (1), the proportions of cetyl trimethyl ammonium bromide, ammonium fluoride, ethyl orthosilicate, ethanol and hydrochloric acid are (400-) < 500mg >) < 700- > 800mg > < 2-3mL > < 90-110mL > < 1.5-3 mL;

mesoporous SiO in step (2) ₂ The proportion of the particles, the total amount of the ethanol and the 3- (methacryloyloxy) propyltrimethoxysilane is (450-500mg) to (50-70mL) to (1-3 mL);

in the step (3), C ═ C surface modified mesoporous SiO ₂ Particles, phosphate buffer, sialic acid, 3-methacrylamidophenylboronic acid, N-isopropylacrylamide, acrylamide, N- (3-aminopropyl) methacrylamide hydrochloride, N, N ' -methylenebisacrylamide, ammonium persulfate, and N, N, N ', N ' -tetramethylethylenediamine in proportions of (450-.

2. The preparation method of the imprinted mesoporous material according to claim 1, comprising the following steps:

(1) mesoporous SiO ₂ Preparation of

Dispersing pore-foaming agents Cetyl Trimethyl Ammonium Bromide (CTAB) and ammonium fluoride in deionized water, slowly adding Tetraethoxysilane (TEOS), reacting for 1-3 h at 70-100 ℃, then repeatedly washing with the deionized water and ethanol, centrifuging and drying to obtain complete SiO ₂ Particles of said SiO ₂ Dispersing the particles in ethanol, adding hydrochloric acid, refluxing for 18-36 h at 70-100 ℃, repeatedly washing and centrifuging by using deionized water and ethanol, and removing the pore-forming agent eluted to prepare the mesoporous SiO ₂ A particle;

(2) mesoporous SiO ₂ Modified by silanization (MSNs)

Mesoporous SiO ₂ The particles are uniformly dispersed in ethanol to form mesoporous SiO ₂ B of the granuleAn alcohol solution, reacting the alcohol solution with 3- (methacryloyloxy) propyltrimethoxysilane (MPTS) and ethanol for 8-18 h at 40-60 ℃, then repeatedly centrifuging and washing with deionized water and ethanol to remove unreacted components, and drying to obtain the C ═ C surface modified mesoporous SiO ₂ A particle;

(3) preparation of sialic acid surface imprinted mesoporous SiO ₂ Particle (MIP-MSNs)

Adding the C ═ C surface modified mesoporous SiO obtained in the previous step into a reaction vessel ₂ The preparation method comprises the steps of carrying out ultrasonic dispersion on particles, Phosphate Buffer Solution (PBS), Sialic Acid (SA), 3-methylacrylamidophenylboronic acid (MAPBA), N-isopropylacrylamide (NIPAM), acrylamide (AAM), N- (3-aminopropyl) methacrylamide hydrochloride and N, N ' -methylene Bisacrylamide (BIS) uniformly, carrying out magnetic stirring at room temperature, introducing argon gas into a reaction system, adding Ammonium Persulfate Solution (APS) and N, N, N ', N ' -tetramethylethylenediamine solution (TMEDA) into the reaction system, placing the reaction container in a water bath at 35-40 ℃ for reaction for 12-36 h, carrying out centrifugal collection and dialysis, carrying out centrifugal collection on the particles again, and obtaining mesoporous SiO (SiO) with sialic acid imprinted on the surface ₂ Particles (MIP-MSNs);

in the step (1), the proportions of cetyl trimethyl ammonium bromide, ammonium fluoride, ethyl orthosilicate, ethanol and hydrochloric acid are (400-) < 500mg >) < 700- > 800mg > < 2-3mL > < 90-110mL > < 1.5-3 mL;

mesoporous SiO in step (2) ₂ The proportion of the particles, the total amount of ethanol and the 3- (methacryloyloxy) propyltrimethoxysilane is (450-500mg): (50-70mL): 1-3 mL);

in the step (3), C ═ C surface modified mesoporous SiO ₂ The ratio of particles, phosphate buffer, sialic acid, 3-methacrylamidophenylboronic acid, N-isopropylacrylamide, acrylamide, N- (3-aminopropyl) methacrylamide hydrochloride, N, N ' -methylenebisacrylamide, ammonium persulfate, N, N, N ', N ' -tetramethylethylenediamine was (450-500mg), (25-35mL), (15-20mg), (10-13mg), (80-85mg), (23-27mg), (10-12mg), (6-10mg), (45-55 μ L) and (95-105 μ L).

3. The preparation method of the imprinted mesoporous material according to claim 2, wherein the magnetic stirring time in the step (3) is 2-6 h.

4. The preparation method of the imprinted mesoporous material according to claim 2, wherein the argon gas is introduced in the step (3) for 20-60 min.

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