CN114601817B

CN114601817B - Degradable hollow organic silicon nano particle and preparation method and application thereof

Info

Publication number: CN114601817B
Application number: CN202210133413.1A
Authority: CN
Inventors: 刘颖; 王紫瑶; 陈春英; 赵宇亮
Original assignee: National Center for Nanosccience and Technology China
Current assignee: National Center for Nanosccience and Technology China
Priority date: 2022-02-11
Filing date: 2022-02-11
Publication date: 2023-01-10
Anticipated expiration: 2042-02-11
Also published as: CN114601817A

Abstract

The invention relates to the field of biological materials, in particular to degradable hollow organic silicon nano particles and a preparation method and application thereof. The degradable hollow organic silicon nano particle comprises a shell layer, wherein the shell layer is a composite layer of organic silicon containing disulfide bonds and inorganic silicon, the composite layer is prepared by taking BTES as an organic silicon source and TEOS as an inorganic silicon source, and the volume ratio of BTES to TEOS is (9-1). The degradable hollow organic silicon provided by the invention effectively loads the drug/antigen by utilizing the shell layer hollow structure, so that the use amount of the carrier is effectively reduced, the use amount of useless carrier components is effectively reduced while delivering goods, redundant glutathione in tumor cells can be consumed, the degradable characteristic of the degradable hollow organic silicon provides guarantee for safe delivery, and the safety is improved while effectively killing the tumor cells.

Description

Degradable hollow organic silicon nano particle and preparation method and application thereof

Technical Field

The invention relates to the field of biological materials, in particular to degradable hollow organic silicon nano particles and a preparation method and application thereof.

Background

Worldwide, the incidence and mortality of cancer have increased year by year, and therefore, the focus of research on how to prevent cancer and how to take effective treatment for cancer is currently important.

The current treatment for cancer depends mainly on the advanced stage of the patient, and the corresponding selection is made according to the physical condition and the tumor condition, including but not limited to radiotherapy, chemotherapy, surgical excision, small molecule targeted therapy, immunotherapy, etc. Currently, clinical treatment generally has great side effects, and the survival time of patients is improved and the prognosis is poor, so the research on cancer treatment at present mainly focuses on the high-efficiency and reasonable delivery of medicines except the molecular mechanism of cancer pathogenesis, the design and synthesis of new medicines, and the prediction and discovery of new molecular targets. Silicon-based related materials are used as common materials for drug delivery due to their characteristics of easy synthesis, convenient modification, and good biocompatibility.

Gas therapy is one of the emerging fields in recent years. The presence of a series of specific gas molecules such as NO, CO, H in the organism ₂ And S, which can coordinate with transition metal to act as messenger to transmit signal to regulate and control physiological process in human body, such as nervous system, cardiovascular system, immune system, etc. Researches show that NO and CO have selective anticancer effect, namely, energy in cancer cells is consumed through the Woberg effect to accelerate apoptosis of the cancer cells and inhibit proliferation of the tumor cells so as to achieve the aim of inhibiting growth and development of the tumor. The selective anticancer of the gases is beneficial to reducing the side effect of treatment and reducing the use of chemotherapy drugs. However, these are too high in blood to cause toxicity, so that a safe and effective carrier is required to prevent its release without temporal and spatial mismatch, and achieve a temporally and spatially proper delivery.

Tumor vaccines have also been developed in recent years by delivering tumor antigens such as tumor cells, tumor-associated proteins, tumor-associated polypeptides, and tumor-associated nucleic acids to patients to stimulate the patients' own immune system, thereby inhibiting tumor growth and progression. The current tumor vaccines are mainly divided into universal tumor vaccines and personalized tumor vaccines, wherein the universal tumor vaccines mainly adopt tumor-associated antigens, namely, associated proteins which exist in a body and are only highly expressed on the surface of tumor cells; and the personalized tumor vaccine selects related protein specifically expressed by tumor cells as antigen. Activation of immunity by payload delivery of antigens is also an important approach to tumor therapy.

However, the silicon-based materials for drug delivery at present have the defects of low degradability and low drug-loading rate, and the silicon-based materials mainly comprise silicon dioxide and silicon nanoparticles derived from the silicon dioxide, and the main body of the silicon-based materials only exists as a carrier, so that the silicon-based materials have the possibility of low loading capacity, non-degradability and potential toxicity; there have also been some studies using silicones to reduce the potential for their potential toxicity using their reduction-responsive cleavage as a carrier but their responsiveness being too sensitive may cause premature release of the drug; and while direct inhalation of gases such as NO, CO can cause excessive gas concentration in blood to cause poisoning, antigen delivery also requires a responsive carrier to increase the efficiency of immune response.

Disclosure of Invention

In order to solve the technical problems, the invention provides a degradable hollow organic silicon nano particle and a preparation method and application thereof. The degradable hollow organic silicon provided by the invention effectively loads the drug/antigen by utilizing the shell layer hollow structure, so that the carrier usage amount is effectively reduced, the reduction responsiveness of the organic silicon is utilized to realize the controllable release of the drug and simultaneously consume the excessive glutathione in the tumor cells, the inorganic silicon component of the degradable hollow organic silicon provides certain rigidity for the structure, the integral selectivity can be improved, and the premature release of the drug is avoided.

In a first aspect, the degradable hollow organosilicon nanoparticle provided by the invention comprises a shell layer, wherein the shell layer is a composite layer of organosilicon containing disulfide bonds and inorganic silicon, the composite layer is prepared by taking BTES as an organic silicon source and TEOS as an inorganic silicon source, and the volume ratio of BTES to TEOS is 9-1.

The degradable silicon nano particle with bioactivity provided by the invention is a composite layer of disulfide bond-containing organic silicon and inorganic silicon with a hollow structure. The composite layer is good in water dispersibility and high in cargo load, is better used for delivering gas prodrugs and antigens and adjuvants, and can control the degradation performance of a carrier. Meanwhile, the degradable silicon nano particles effectively reduce the using amount of carriers by efficiently loading the drugs/antigens by utilizing the hollow structure of the shell layer, effectively reduce the using amount of useless carrier components while delivering cargos, consume redundant glutathione in tumor cells, and keep the hollow rigidity of inorganic components from the structure to avoid premature release of the inorganic components. The design of the organic-inorganic hybrid shell can better realize the controllable release of the drug/antigen, provides guarantee for safer delivery, and improves the safety while effectively killing tumor cells. The silicon source of the shell layer of the degradable hollow organic silicon nano particle adopts a mixture of an organic silicon source and an inorganic silicon source to adjust the degradation rate of the carrier so as to realize the release of the cargo.

According to the invention, the volume ratio of the organic silicon source to the inorganic silicon source in shell deposition is 9-1. For example, the ratio of 9. According to the invention, the BTES and the TEOS in a specific proportion can better play a synergistic effect between the BTES and the TEOS, so that the effect is better, the degradation rate of the carrier can be better adjusted to release the goods, and the shell layer performance is optimal.

Further preferably, the drug-containing shell further comprises at least one of a drug, a tumor antigen and an adjuvant loaded in the shell; preferably, the drug is at least one of a chemotherapeutic drug, a carbon monoxide prodrug and a nitric oxide prodrug; more preferably the carbon monoxide prodrug Mn ₂ (CO) ₁₀ 。

In order to further improve the cell-entering effect of the medicine-carrying organic silicon nano-particles, the invention optimizes the structural parameters of the nano-particles, and the thickness of the shell layer is 20-40 nm; and/or the particle size of the degradable hollow organic silicon nano particle is 180-240 nm.

It is further preferred that the degradable hollow silicone nanoparticles have a particle size of 150-200 nm, such as 150nm, 155nm, 160nm, 165nm, 170nm, 175nm, 180nm, 185nm, 190nm, 195nm or 200nm and specific values therebetween, for reasons of brevity and clarity, the present invention is not exhaustive of the specific values included in the ranges, and a more preferred particle size of about 200nm is preferred.

In a second aspect, the invention provides a preparation method of degradable hollow organosilicon nanoparticles (carriers). The method can adopt a hard template method, takes silicon dioxide spheres with super-good dispersibility as a template, takes an organic silicon source BTES and an inorganic silicon source TEOS with a certain proportion as silicon sources, hydrolyzes and deposits a layer of composite structure of inorganic silicon and organic silicon containing disulfide bonds on the surface of the template to be used as a shell layer, and finally selectively etches away the silicon dioxide spheres inside. Specifically, the preparation method of the degradable hollow organosilicon nano particle comprises the following steps:

1) Using ammonia water as a catalyst, using TEOS as an inorganic silicon source, hydrolyzing in an organic solvent-water composite solvent system under the stirring condition, and washing and resuspending to obtain a silicon dioxide core;

2) Mixing the silicon dioxide core in the step 1) with a uniform aqueous solution system containing CTAC and TEA, and stirring to obtain a uniform dispersion liquid containing the silicon dioxide core;

3) Slowly dripping a mixed solution of TEOS and BTES into the uniform dispersion liquid containing the silicon dioxide cores in the step 2) at a high temperature to react for a period of time, washing and resuspending the obtained precipitate, and obtaining core-shell composite silicon nanoparticles;

4) Selectively etching the core-shell composite silicon nano particles in the step 3) by using an etching agent to obtain coarse hOS;

5) Extracting the crude hOS in the step 4) by using a methanol solution of salt to obtain hOS nano particles without the surfactant; and, optionally

6) Loading the hOS nano-particles in the step 5) with drugs.

The method for preparing the degradable hollow organic silicon nano particles can improve the transmission quantity of the medicine/antigen, enhance the curative effect of the medicine, reduce the side effect of the medicine and enhance the immunity.

Preferably, according to the preparation method of the degradable hollow organosilicon nanoparticle, in step 1), the organic solvent is chloroform, methanol, ethanol, dichloromethane or tetrahydrofuran; ethanol is preferred.

The invention can make the effect of the prepared nano particles better by adopting the optimized treatment condition.

Further preferably, in the step 1), the stirring is magnetic stirring.

Further preferably, in the step 1), the volume ratio of the organic solvent to water to ammonia water to TEOS is 33-40; preferably, the ratio of 37.

Further preferably, in step 1), the washing and resuspending steps are: centrifuging at 7000-9000 rpm for 5-10 min; adding 5-20 mL of ethanol into the centrifuged precipitate, uniformly dispersing, centrifuging, and repeating for multiple times; adding 5-20 mL of water into the precipitate, dispersing, centrifuging, repeating for multiple times, and then suspending in 10-50 mL of water; preferably, centrifugation is carried out for 8min at 8000 rpm; adding 10mL of ethanol into the centrifuged precipitate, uniformly dispersing, centrifuging, and repeating for three times; then, 10mL of triple water was added to the pellet, dispersed, centrifuged, repeated three times, and finally resuspended in 30mL of triple water.

According to a preferred embodiment of the present invention, the silica spheres have a good dispersibility, the silica spheres have a particle size of 100nm to 400nm, which may be, for example, about 100nm, about 200nm, about 300nm or about 400nm, and specific values therebetween, which are not exhaustive and included in the range, preferably about 200nm, for reasons of brevity and conciseness.

Further preferably, in the step 2), the uniform aqueous solution system is CTAC (CTAC solution), TEA and water in a proportion of 1-3 g: 0.03-0.05 g: 80-100 mL of mixed homogeneous solution; the concentration of CTAC is preferably 22-27 wt%; further preferably, the mass ratio of the CTAC and the TEA is 2. CTAC (25 wt%), 2g; TEA,0.04g; three times water, 90mL.

Further preferably, the silica core is 30mL of the SiO2 resuspension obtained in step 1).

Further preferably, in step 2), the stirring is magnetic stirring, and preferably, the stirring time is 2 hours.

Preferably, according to the preparation method of the degradable hollow organosilicon nanoparticle, in step 3), the volume ratio of BTES to TEOS is 9.

Further preferably, in step 3), the high temperature is 75 to 90 ℃, preferably 80 ℃.

More preferably, in the step 3), the stirring is magnetic stirring.

More preferably, in the step 3), the slow dropping is performed at 10 to 15 drops/min.

More preferably, in step 3), the reaction time is 1 to 3 hours, preferably 2 hours.

Further preferably, in step 3), the washing reagent is ethanol.

Further preferably, in step 3), the washing conditions are as follows: firstly, the centrifugation condition is 8000rpm,8min; and adding 10mL of ethanol into the centrifuged precipitate, uniformly dispersing, centrifuging and repeating twice. Then 10mL of triple water was added to the pellet, dispersed, centrifuged, repeated three times, and finally resuspended in 30mL of triple water.

Preferably, according to the method for preparing the degradable hollow organosilicon nanoparticles, in the step 4), the etchant is selected from one or more of sodium hydroxide, potassium hydroxide, ammonia water, sodium bicarbonate, ammonium carbonate and sodium carbonate, and sodium carbonate is preferred.

More preferably, in the step 4), the concentration of the etchant is 0.01 to 0.1M.

In the invention, the etching agent is an alkaline reagent, and is selected from one or more of sodium hydroxide, potassium hydroxide, ammonia water, sodium bicarbonate, ammonium carbonate and sodium carbonate, and can also be other substances with alkalinity, and sodium carbonate is preferred. The concentration of the etchant may be 0.01M to 0.1M, for example, 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M or 0.1M and specific values therebetween, preferably a sodium carbonate solution concentration of 0.05M. The etching time is 2h to 24h, and may be, for example, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h or 24h and specific values therebetween, preferably 10h. The degradable hollow organic silicon nano particles synthesized according to the proportion have the advantages of good stability, good dispersibility and large loading capacity, and have great potential as a delivery carrier of medicines/antigens.

Further preferably, in the step 4), the method specifically includes:

a. preparing a sodium carbonate solution;

b. carrying out ultrasonic treatment on the MOS solution obtained in the step 3);

c. diluting the sodium carbonate solution in the step a;

d. mixing the MOS solution subjected to ultrasonic treatment with the diluted sodium carbonate solution;

e. c, stirring the mixed solution in the step c for reaction;

f. the reaction product was washed by centrifugation.

Preferably, in step a, the concentration of the sodium carbonate solution is 1M.

And/or in the step b, the ultrasonic time is 10-20 min.

And/or, in the step b, the volume of the MOS solution is 10mL.

And/or in step c, the final concentration of the diluted sodium carbonate solution is 0.01-0.1M, preferably 0.05M.

And/or, in step e, the stirring is magnetic stirring.

And/or, in step e, the temperature of the stirring is 25 ℃.

And/or in the step e, the reaction time is 10h.

And/or, in the step f, the centrifugal washing condition is 13000rpm,10min.

Further preferably, in step 5), the methanol solution of the salt is a methanol solution of NaCl.

Further preferably, the step 5) specifically comprises the following steps: mixing 10mL of MOS crude solution and a NaCl methanol solution with the mass fraction of 1.5% in an equal volume manner on a shaking table, incubating for 10h, centrifuging at 13000rpm for 10min to obtain a hOS precipitate, suspending in 10mL of methanol, mixing the hOS precipitate and a NaCl methanol solution with the mass fraction of 1.5% in an equal volume manner on the shaking table, incubating for 10h, washing twice with methanol and twice with water under the same centrifugation condition, dispersing in 10mL of water, and freeze-drying to obtain hOS powder.

Preferably, according to the preparation method of the degradable hollow silicone nanoparticle, in the step 6), the preparation method specifically includes:

A. dissolving the hOS nano particles obtained after freeze-drying in an organic solvent to obtain a first dispersion liquid;

B. carbon monoxide prodrug Mn ₂ (CO) ₁₀ Dissolving in the organic solvent to obtain a second solution;

C. mixing the first solution and the second solution to obtain a third dispersion liquid;

D. stirring the third dispersion liquid at the temperature of 2-6 ℃;

E. centrifuging the stirred third dispersion of step D, and washing with methanol and water in sequence.

Further preferably, steps B-D are performed under exclusion of light.

More preferably, in the step a, the concentration of the hOS nanoparticles in the first dispersion is 0.1 to 2mg/mL; for example, the concentration may be 0.1mg/mL, 0.2mg/mL, 0.6mg/mL, 1mg/mL, 1.5mg/mL, 1.6mg/mL, 1.8mg/mL, 1.9mg/mL, or 2mg/mL, but for reasons of brevity and brevity, the present invention is not exhaustive, and the concentration is most preferably 1mg/mL, and too low a concentration will affect the drug loading efficiency.

More preferably, in steps a and B, the organic solvent is any one of methanol and ethanol, preferably methanol.

Further preferably, in step B, the carbon monoxide prodrug Mn ₂ (CO) ₁₀ The concentration of (a) is 0.1-2 mg/mL; for example, it may be 0.1mg/mL, 0.2mg/mL, 0.6mg/mL, 1mg/mL, 1.5mg/mL, 1.6mg/mL, 1.8mg/mL, 1.9mg/mL, or 2mg/mL, depending on the space and the originFor the sake of brevity, the invention is not intended to be exhaustive of the specific points included in the ranges, with a concentration of 2mg/mL being most preferred.

More preferably, in step C, the mixing is performed by sonication, and the volume ratio of the first dispersion to the second dispersion is 0.1; for example, it can be 0.1.

More preferably, in step D, the mixture is stirred at 3-5 ℃ for 12-24 h.

Further preferably, the stirring time of the third dispersion at 4 ℃ is 12h to 24h, for example, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h or 24h, but is not limited to space and for the sake of brevity, the present invention is not exhaustive, and the specific point value included in the range is preferably 12h.

Further preferably, in the step E, the rotating speed of the centrifugation is 8000-12000 rpm, and the centrifugation time is 5-20 min; for example, it may be 8000rpm, 9000rpm, 9100rpm, 9300rpm, 10000rpm, 10500rpm, 11000rpm or 13000rpm, preferably 13000rpm; the centrifugation time is preferably 5min to 20min, for example 5min, 8min, 12min, 15min or 20min, preferably 10min. The resuspension volume was 1mL.

The degradable nanoparticles with biological activity prepared by the invention can enhance the dispersibility of the loaded drug in water and enhance the toxicity of the loaded drug to A549 lung cancer cells. The degradable hollow organosilicon nano particle has better safety and has no obvious influence on the growth of normal lung epithelial cells B2B.

In a third aspect, the invention provides an application of the degradable hollow organic silicon nano particle or the preparation method of the degradable hollow organic silicon nano particle in preparation of a medicament for inhibiting tumor growth of A549 lung cancer cells.

The invention has the beneficial effects that: the degradable hollow organosilicon nano particle mainly takes a silicon dioxide ball with super-good dispersibility as a template, the composite coating of organosilicon inorganic silicon is deposited on the surface of the silica dioxide ball by a hydrolysis method, the silica dioxide ball has better dispersibility in water and alcohol solvents, the loading of drugs with poor water solubility is facilitated, the degradation capability of the material is adjusted by using the characteristics of organic and inorganic composite, so that the space-time coupled drug/antigen release is achieved, the degradable hollow organosilicon nano particle has the potential that different modifications can be carried out, the drug with poor water solubility is carried, and goods are highly loaded, so that the aim of effectively enhancing tumor treatment/tumor immunity is fulfilled.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 SiO prepared in example 1 ₂ 、SiO ₂ Transmission electron micrographs of @ OS and hOS;

FIG. 2 is a diagram showing the ultra-fine dispersion SiO prepared in step (1) of example 1 ₂ A transmission electron microscope image;

FIG. 3 is a high power transmission electron micrograph of hOS prepared in example 1;

FIG. 4 is a Fourier transform infrared spectrum of hOS and CTAC prepared in example 1;

FIG. 5 is a scanning electron micrograph of hOS prepared in example 1;

FIG. 6 is a spectrum of hOS prepared in example 1;

FIG. 7 is a spectrum of hOS prepared in example 1;

FIG. 8 is a spectrum of hOS prepared in example 1;

FIG. 9 is a spectrum of hOS prepared in example 1;

FIG. 10 shows Mn obtained in example 2 ₂ (CO) ₁₀ Standard plots of supernatants for @ hOS;

FIG. 11 is an electron micrograph of nanoparticles prepared in comparative example 1;

FIG. 12 is an electron micrograph of nanoparticles prepared in comparative example 2;

FIG. 13 is an electron micrograph of nanoparticles prepared in comparative example 3;

FIG. 14 is an A549 lung cancer cell viability map obtained in example 3;

FIG. 15 is a B2B normal lung epithelial cell viability map obtained in example 4;

FIG. 16 is a B2B normal lung epithelial cell viability map obtained in example 5;

fig. 17 is a graph showing the viability of a549 lung cancer cells obtained in example 6.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and the following embodiments are used for illustrating the present invention and are not intended to limit the scope of the present invention. The examples do not specify particular techniques or conditions, and are to be construed in accordance with the description of the art in the literature or with the specification of the product. The reagents or instruments used are conventional products available from normal commercial vendors, not indicated by the manufacturer.

In the invention, english which is partially abbreviated is called as follows:

hOS hollow organic silica nanoparticles；

TEOS Tetraethoxysilane；

BTES Bis[3-(triethoxysilyl)propyl]tetrasulfide；

SiO ₂ @OS organic silica coated SiO ₂ 。

example 1

In this example, the preparation of ultra-good dispersibility by the following method specifically includes the steps of:

(1)SiO ₂ and (3) synthesis of a core: mixing 37mL of absolute ethyl alcohol, 5mL of ultrapure water and 1mL of ammonia water (25% -28%), stirring for 20min in a constant-temperature water bath at 25 ℃ at the rotating speed of 800rpm, and then uniformly stirring3mL TEOS was added dropwise. After the completion of the dropwise addition, the rotation speed was adjusted to 400rpm for 1 hour. Centrifuging at 8000rpm for 8min after the reaction is finished, discarding a supernatant, washing with ethanol for three times under the same centrifugation condition, washing with water for three times, and dispersing a white precipitate product in 30mL of ultrapure water;

(2)SiO ₂ synthesis of @ OS: a250 mL round bottom flask was charged with 2g CTAC (25%), 0.04g triethanolamine and 90mL ultrapure water. Mix for 2h at 800 rpm. Then 30mL of SiO prepared in the previous step was added ₂ The solution was stirred at the same speed for an additional 2h. Then 0.5mL BTES +0.5mL TEOS mixed solution is dropped under the condition of oil bath at 80 ℃ and stirred for 2h at the temperature. And centrifuging after the product is cooled. The obtained SiO ₂ The @ OS product was centrifuged at 8000rpm,8 min. Centrifuging to obtain precipitate, washing with ethanol under the same centrifugation condition for three times, washing with water for three times, and dispersing in 30mL of ultrapure water to obtain MOS solution;

(3) Synthesis of crude hOS: 5.21mL 1M sodium carbonate solution is prepared for standby. The 10mL of MOS solution from the previous step was sonicated for 15min to disperse uniformly. Ultrapure water and 5.21mL of 1M sodium carbonate solution were added to a 250mL round-bottom flask and the solution was mixed uniformly at 400rpm to obtain a 0.05M sodium carbonate solution. After the two solutions are respectively dispersed uniformly, 10mL of MOS solution is added into the sodium carbonate solution. The reaction is carried out for 10h at 25 ℃ under the same rotating speed condition. Centrifuging the product at 13000rpm for 10min after the reaction is finished, washing the obtained precipitate with ultrapure water for three times, and dispersing the precipitate in 10mL of methanol to obtain a crude hOS solution;

(4) Removal of active agent: mixing 10mL of crude hOS solution and a NaCl methanol solution with the mass fraction of 1.5% in an equal volume manner on a shaking table, incubating for 10h, centrifuging at 13000rpm for 10min to obtain hOS precipitate, suspending in 10mL of methanol, mixing the hOS precipitate and a NaCl methanol solution with the mass fraction of 1.5% in an equal volume manner on the shaking table, incubating for 10h, washing twice with methanol under the same centrifugation condition, washing twice with water, dispersing in 10mL of water, and freeze-drying to obtain hOS powder.

Transmission electron microscope Hitachi HT7700 for SiO ₂ 、SiO ₂ @ OS, hOS, as in FIG. 1.

SiO by transmission electron microscope Hitachi HT7700 ₂ Is characterized in the size of the figure 2.

The mesoporous structure of hOS was characterized by a transmission electron microscope Hitachi HT7700, as shown in fig. 3, the particle size of hOS was about 200nm, and the thickness of the mesoporous layer was about 25 nm.

hOS was characterized using fourier transform infrared spectroscopy, as shown in figure 4.

The hOS was characterized using scanning electron microscope S4800, as shown in fig. 5.

hOS was characterized by energy spectra, as shown in fig. 6, 7, 8, 9.

Example 2

In this example, the carbon monoxide prodrug Mn was prepared by loading as follows ₂ (CO) ₁₀ The nano-drug specifically comprises the following steps:

(1) Weighing 1mg of synthesized hollow silicon nanoparticles, adding 0.5mL of methanol, and fully dissolving to obtain a solution A;

(2) Weighing 1mg of carbon monoxide prodrug Mn under the condition of keeping out of the sun ₂ (CO) ₁₀ Adding 0.5mL of methanol, and fully dissolving to obtain a solution B;

(3) Under the condition of keeping out of the sun, uniformly mixing the solution A and the solution B in a 2ml brown bottle, and carrying out ultrasonic treatment for 2min to obtain a solution C;

(4) Stirring for 12h at 4 ℃ in a dark condition;

(5) Centrifuging the obtained product at 13000rpm for 10min, washing with methanol for three times, collecting supernatant for measuring load, washing with water again, suspending in water, and freeze-drying for preservation;

method for measuring Mn by using ultraviolet visible light spectrometry ₂ (CO) ₁₀ The amount of supported Mn was measured to prepare Mn ₂ (CO) ₁₀ The concentration of the supernatant was measured by a standard curve method. The measured load rate is 331mg Mn ₂ (CO) ₁₀ G hOS, as shown in FIG. 10.

Comparative example 1 etching of MOS Using sodium hydroxide

The previous steps are identical to (1) and (2) in example 1, only 0.05M sodium carbonate solution is replaced by 0.05M sodium hydroxide, the etching time is 10h, and the rest steps are the same as example 1. The resulting nanoparticles mostly collapsed due to over-etching (see FIG. 11). Comparative example 2 uses a 0.01M sodium bicarbonate concentration to etch OS

The previous steps are all consistent with (1) and (2) in the first embodiment, the concentration of sodium carbonate is 0.01M, the etching time is 10h, and the rest steps are the same as the first embodiment. The obtained nanoparticles are mostly solid particles (as shown in fig. 12).

Comparative example 3 etching of OS using a sodium bicarbonate concentration of 0.02M

The previous steps are all consistent with (1) and (2) in example 1, the concentration of sodium carbonate is 0.02M only, the etching time is 10h, and the rest steps are the same as example 1. The obtained nanoparticles are mostly solid particles with a slight shell (as shown in FIG. 13).

Example 3

In this example, different concentrations of the carbon monoxide-loaded prodrug Mn were performed by the following method ₂ (CO) ₁₀ The nano-drug cytotoxicity detection specifically comprises the following steps:

cells were seeded at 7000 cells/well in 96-well plates and after overnight adherence, contained different concentrations of the carbon monoxide prodrug Mn ₂ (CO) ₁₀ The nanomedicine of (1 and 2 from examples, at concentrations of 12.5. Mu.g/mL, 25. Mu.g/mL, 50. Mu.g/mL, and 100. Mu.g/mL, respectively) was added to the wells and after 24 hours of treatment, the medium was removed; mu.L of cell culture medium containing 10% (by volume) of CCK-8 was added to each well, and after about half an hour the cells in the 96-well plate turned yellow, the absorbance at 450nm was measured on a microplate reader, and 600nm was used as the reference wavelength. After the absorbance value of the blank solution is subtracted from the absorbance value of each group, the corresponding value of each hole is divided by the absorbance value of the control group to be used as the cell activity. Each set was provided with 4 parallel holes.

As shown in fig. 14, compared with a control group, the nano drug carrier prepared in example 1 can significantly kill a549 lung cancer cells.

Example 4

In this embodiment, the following method is used to detect the effect of different concentrations of hOS nanocarriers on normal lung epithelial cells, and specifically includes the following steps:

B2B lung normal epithelial cells were seeded at 7000 cells/well in 96-well plates and after overnight adherence empty nanocarriers containing different concentrations of hOS (from example 1 at concentrations of 62.5 μ g/mL, 125 μ g/mL, 250 μ g/mL, and 250 μ g/mL, respectively) were added to the wells and after 24 hours of treatment the medium was removed; mu.L of cell culture medium containing 10% (volume ratio) of CCK-8 was added to each well, cells in the well plate turned yellow after continued culture in the incubator for about half an hour, absorbance value at 450nm was measured on a microplate reader, and 600nm was used as a reference wavelength. After the absorbance value of the blank solution is subtracted from the absorbance value of each group, the corresponding value of each hole is divided by the absorbance value of the control group to be used as the cell activity. Each set was provided with 4 parallel holes.

The cell viability measurement result is shown in fig. 15, and compared with the control group, the hOS nano empty vector prepared in example 1 has no obvious toxic effect on normal lung epithelial cells B2B, which indicates that the prepared nano empty vector has higher safety.

Example 5

In this example, the detection of the longer-term effect of high-concentration hOS nanocarriers on normal lung epithelial cells was performed by the following method, which specifically includes the following steps:

inoculating B2B lung normal epithelial cells into a 96-well plate at 7000 cells/well density, and after the cells adhere to the wall overnight, mixing the cells with an empty nano-carrier of hOS and a carbon monoxide prodrug Mn ₂ (CO) ₁₀ Carrying a carbon monoxide prodrug Mn ₂ (CO) ₁₀ hOS (from example 1, at a concentration of 500. Mu.g/mL) was added to the wells and after 24 and 48 hours of treatment, respectively, the medium was removed; mu.L of cell culture medium containing 10% (volume ratio) of CCK-8 was added to each well, cells in the well plate turned yellow after continued culture in the incubator for about half an hour, absorbance value at 450nm was measured on a microplate reader, and 600nm was used as a reference wavelength. After the absorbance value of the blank solution is subtracted from the absorbance value of each group, the corresponding value of each hole is divided by the absorbance value of the control group to be used as the cell activity. Each set was provided with 4 parallel wells.

The cell viability measurement result is shown in fig. 16, compared with the control group, the hOS nano empty vector prepared in example 1 has no obvious toxic effect on normal lung epithelial cell B2B after 24 hours and 48 hours of culture at the higher concentration used, which indicates that the prepared nano empty vector has higher safety.

Example 6

In this embodiment, the detection of the effectiveness of the hOS nanocarrier is performed by the following method, which specifically includes the following steps:

a549 Lung cancer cells were seeded at 7000 cells/well into 96-well plates and after overnight adherence, empty nanocarriers of hOS (from example 1 at a concentration of 100. Mu.g/mL) were loaded with free carbon monoxide prodrug Mn ₂ (CO) ₁₀ (at a concentration of 100 ug/mL), and the loaded carbon monoxide prodrug Mn ₂ (CO) ₁₀ hOS of (2), i.e. Mn ₂ (CO) ₁₀ @ hOS (from example 2, at a concentration of 100 ug/mL) was added to the wells and after 24 hours of treatment the medium was removed; mu.L of cell culture medium containing 10% (volume ratio) of CCK-8 was added to each well, cells in the well plate turned yellow after continued culture in the incubator for about half an hour, absorbance value at 450nm was measured on a microplate reader, and 600nm was used as a reference wavelength. After the absorbance value of the blank solution is subtracted from the absorbance value of each group, the corresponding value of each hole is divided by the absorbance value of the control group to be used as the cell activity. Each set was provided with 4 parallel holes.

Cell viability assay results are shown in FIG. 17, comparing Mn prepared in example 2 with control ₂ (CO) ₁₀ The @ hOS nano-drug can obviously improve Mn ₂ (CO) ₁₀ Stability and dispersibility of (1), enhanced Mn ₂ (CO) ₁₀ Cytotoxicity to a549 lung cancer cells.

Although the invention has been described in detail with respect to the general description and the specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A degradable hollow organic silicon nanoparticle is characterized by comprising a shell, wherein the shell is a composite layer of organic silicon containing disulfide bonds and inorganic silicon, the composite layer is prepared by taking BTES as an organic silicon source and TEOS as an inorganic silicon source, and the volume ratio of BTES to TEOS is (9) - (1);

the preparation method of the degradable hollow organic silicon nano particle comprises the following steps:

1) Hydrolyzing in an organic solvent-water composite solvent system under the stirring condition by taking ammonia water as a catalyst and TEOS as an inorganic silicon source, and washing and resuspending to obtain silicon dioxide cores;

4) Selectively etching the core-shell composite silicon nanoparticles in the step 3) by using an etching agent to obtain coarse hOS nanoparticles;

5) Extracting the crude hOS in the step 4) by using a methanol solution of salt to obtain hOS nano particles without the surfactant;

6) Carrying out drug loading on the hOS nano particles in the step 5);

in the step 1), the organic solvent is chloroform, methanol, ethanol, dichloromethane or tetrahydrofuran; in the step 3), the high temperature is 75 to 90 ℃; in the step 3), the washing reagent is ethanol; in the step 4), the etching agent is sodium carbonate.

2. The degradable hollow silicone nanoparticle of claim 1, further comprising a drug loaded within the shell; the drug is a carbon monoxide prodrug.

3. The degradable hollow silicone nanoparticle of claim 2, wherein the degradable hollow silicone nanoparticle is characterized byThe drug being a carbon monoxide prodrug Mn ₂ (CO) ₁₀ 。

4. The degradable hollow organosilicon nanoparticle as claimed in any one of claims 1 to 3, wherein the thickness of the shell layer is 20 to 40nm; and/or the particle size of the degradable hollow organic silicon nano particle is 180 to 220 nm.

5. The method for preparing degradable hollow silicone nanoparticles according to any one of claims 1-4, characterized by comprising the steps of:

3) Slowly dripping the mixed solution of TEOS and BTES into the uniform dispersion liquid containing the silicon dioxide core in the step 2) at high temperature for reaction for a period of time, washing the obtained precipitate, and carrying out heavy suspension to obtain the core-shell composite silicon nano particles;

4) Selectively etching the core-shell composite silicon nano particles in the step 3) by using an etching agent to obtain coarse hOS nano particles;

6) Carrying out drug loading on the hOS nano particles in the step 5).

6. The preparation method of the degradable hollow organosilicon nanoparticles according to claim 5, characterized in that in step 1), the volume ratio of the organic solvent, water, ammonia water and TEOS is 33 to 40;

and/or in the step 2), the uniform aqueous solution system is prepared by mixing CTAC, TEA and water according to the proportion of 1-3 g:0.03 to 0.05g:80 to 100mL of a mixed uniform solution; the concentration of CTAC is 22 to 27 wt%.

7. The preparation method of the degradable hollow organosilicon nanoparticles according to claim 5, wherein in step 3), the volume ratio of BTES to TEOS is 9; and/or the stirring is magnetic stirring; and/or, the slow dripping is 10-15 drops/min; and/or the reaction time is 1 to 3 hours.

8. The method for preparing degradable hollow silicone nanoparticles according to claim 7, wherein in step 3), the volume ratio of BTES to TEOS is 1.

9. The preparation method of the degradable hollow organosilicon nanoparticles as claimed in claim 5, wherein in the step 4), the concentration of the etching agent is 0.01-0.1M; and/or, in step 5), the methanol solution of the salt is a NaCl methanol solution.

10. The method for preparing degradable hollow silicone nanoparticles according to any one of claims 5-9, wherein the step 6) specifically comprises:

A. dissolving the obtained hOS nano particles in an organic solvent to obtain a first dispersion liquid;

D. the third dispersion liquid is in the range of 2 to 6 ^o Stirring under the condition of C;

E. and D, centrifuging the stirred third dispersion liquid in the step D, and washing the third dispersion liquid with methanol and water in sequence.

11. The method for preparing degradable hollow silicone nanoparticles according to claim 10, wherein steps B-D are performed under light-shielding conditions;

and/or in the step A, the concentration of the hOS nano particles in the first dispersion liquid is 0.1-2 mg/mL;

and/or in the steps A and B, the organic solvent is any one of methanol and ethanol;

and/or, in step B, the carbon monoxide prodrug Mn ₂ (CO) ₁₀ The concentration of (A) is 0.1-2 mg/mL;

and/or in the step C, the mixing mode is ultrasonic, and the volume ratio of the first dispersion liquid to the second dispersion liquid is 0.1;

and/or in the step D, the temperature is within 3 to 5 ^o Stirring for 12-24 h under C;

and/or in the step E, the rotating speed of the centrifugation is 8000-12000 rpm, and the centrifugation time is 5-20 min.

12. The use of the degradable hollow silicone nanoparticle of any one of claims 1 to 4 or the degradable hollow silicone nanoparticle obtained by the preparation method of the degradable hollow silicone nanoparticle of any one of claims 5 to 11 in the preparation of a medicament for inhibiting the growth of a549 lung cancer cell tumors.