CN113768903A

CN113768903A - Brown algae oligosaccharide modified aminated mesoporous silica nanoparticle

Info

Publication number: CN113768903A
Application number: CN202111060301.XA
Authority: CN
Inventors: 韩榕; 欧阳小琨; 黄依如; 范丽红
Original assignee: Zhejiang Ocean University ZJOU
Current assignee: Zhejiang Ocean University ZJOU
Priority date: 2021-09-10
Filing date: 2021-09-10
Publication date: 2021-12-10
Anticipated expiration: 2041-09-10
Also published as: CN113768903B

Abstract

The invention provides a preparation method of brown alga oligosaccharide modified aminated mesoporous silica nanoparticlesAminated mesoporous silica (MSN-NH)₂) Then, the brown algae oligosaccharide is functionally modified to a nucleus MSN-NH with positive electricity₂To obtain MSN-NH₂AOS core-shell nanoparticles, whose performance in loading and transporting curcumin was studied. MSN-NH of the invention₂the-Cur-AOS has higher drug transfer efficiency, can realize pH sensitive responsive drug release, intelligently release drugs in the tumor cells with partial acidity, and has good killing effect on the tumor cells.

Description

Brown algae oligosaccharide modified aminated mesoporous silica nanoparticle

Technical Field

The invention relates to a mesoporous silica nanoparticle, in particular to a brown alga oligosaccharide aminated mesoporous silica nanoparticle sensitive to delivery of curcumin pH.

Technical Field

Curcumin (Curcumin, Cur) is a plant-derived polyphenolic compound extracted from turmeric and has anti-tumor, antioxidant, anti-amyloid and anti-inflammatory properties. However, curcumin has the disadvantages of strong hydrophobicity, easy oxidation in vitro, poor stability, low oral bioavailability and the like, and influences the clinical application of curcumin. In previous studies, scientists have solved such problems by encapsulating Cur in micelles, liposomes, nanocarriers, etc. The pH response type drug delivery system can effectively protect normal cells of a body, and simultaneously deliver the drug to a tumor part in a slightly acidic environment to kill tumor cells, thereby effectively reducing the side effect of the drug. Therefore, the focus of research is to develop pH sensitive systems as effective curcumin delivery vehicles.

Disclosure of Invention

In order to realize curcumin pH response release, the inventor prepares a functional nano drug-loaded particle of alginate-oligosaccharide (AOS) -modified Mesoporous Silica (MSN) through long-term research, improves the stability and bioavailability of curcumin, realizes fixed-point drug release at a tumor part, and simultaneously, the nano drug delivery system has good external release characteristic, cell uptake and safety.

The preparation method of the functional nano drug-loaded particles of the brown Alginate Oligosaccharide (AOS) -modified Mesoporous Silica (MSN) comprises the following steps:

1) preparation of Mesoporous Silica (MSN)

Dissolving hexadecyl trimethyl ammonium chloride in deionized water, dropwise adding triethanolamine, stirring at 95 ℃ for 1 hour, slowly adding tetraethoxysilane into the mixed solution, reacting for 1 hour, washing the product with the deionized water and ethanol for three times respectively, then carrying out vacuum drying at-60 ℃, and then calcining in a muffle furnace at 550 ℃ for 6 hours to remove a template, thereby obtaining a product MSN;

2) preparation of aminated MSN nanoparticles

Reacting MSN and 3-aminopropyltriethoxysilane in toluene at 60 deg.C for 24 hr, washing with ethanol and water alternately after reaction to obtain product MSN-NH₂；

3) Preparation of alginate oligosaccharide coated aminated mesoporous silica nanoparticles

Preparing 1mg/mL curcumin solution with anhydrous ethanol, adding phosphoric acid buffer solution and MSN-NH into curcumin solution₂Stirring at room temperature in dark place for 12 hours; obtaining the MSN-NH loaded with curcumin (Cur)₂A solution;

preparing 1mg/mL brown algae oligosaccharide solution by using ultrapure water, taking the brown algae oligosaccharide solution, adding N-hydroxysuccinimide (NHS) and 1-ethyl-carbodiimide (EDC), and stirring for 4 hours in the dark to activate carboxyl, thereby obtaining the activated brown algae oligosaccharide solution;

loading curcumin (Cur) loaded MSN-NH₂The solution is dripped into the activated brown algae oligosaccharide solution, stirred at room temperature in the dark for 12 hours, and centrifuged at 8000r/min for 10min to obtain the brown algae oligosaccharide coated aminated mesoporous silica nano particle MSN-NH₂-Cur-AOS。

Preferably, the weight-to-volume ratio g/mL of the hexadecyl trimethyl ammonium chloride to the deionized water in the step 1) is 1: 10.

Preferably, the weight-volume ratio g/mL of the hexadecyl trimethyl ammonium chloride to the triethanolamine in the step 1) is 25: 4.

Preferably, the weight-volume ratio g/mL of the hexadecyl trimethyl ammonium chloride to the tetraethoxysilane in the step 1) is 4: 3.

Preferably, the weight-to-volume ratio g/mL of the MSN to the 3-aminopropyltriethoxysilane in the step 2) is 1: 20; the volume ratio of the 3-aminopropyltriethoxysilane to the toluene in the step 2) is 1: 5.

Preferably, the pH value of the phosphoric acid buffer solution in the step 3) is 7.4.

Preferably, the volume ratio of the curcumin solution to the phosphoric acid buffer solution in the step 3) is 1:10, said MSN-NH₂The weight-to-volume ratio of mg/ml to curcumin solution was 20: 1.

Preferably, the weight-to-volume ratio mg/ml of the NHS to the alginate-derived oligosaccharide solution in the step 3) is 5: 2.

Preferably, the weight ratio of NHS to 1-ethyl-carbonyldiimine in said step 3) is 1: 2.

Preferably, the step 3) is carried out by loading MSN-NH of curcumin (Cur)₂The volume ratio of the solution to the activated alginate-derived oligosaccharide solution is 1: 1.

The research adjusts the mass ratio of the alginate-derived oligosaccharide (AOS) to the Mesoporous Silica (MSN) by the interaction of the amino group of the aminated mesoporous silica and the carboxyl ion of the alginate-derived oligosaccharide, so as to obtain the nano drug-loaded particles with the optimal ratio. MSN-NH of the invention₂the-Cur-AOS has higher drug transfer efficiency, can realize pH sensitive responsive drug release, intelligently release drugs in the tumor cells with partial acidity, and has good killing effect on the tumor cells.

Drawings

FIG. 1 is a schematic diagram of the preparation and release of MSN-NH2-Cur-AOS nanoparticles.

FIG. 2 is a transmission electron microscope image of MSN nanoparticles, and a graph showing changes in particle size, potential and PDI of the MSN nanoparticles;

wherein 2a is MSN-1 transmission electron microscope picture, 2b is MSN-2 transmission electron microscope picture, 2c is MSN transmission electron microscope picture, 2d is 1 MSN-1, 2 is MSN-2, 3 is MSN.

FIG. 3a shows MSN-NH prepared in different ratios₂The particle size and zeta potential of AOS, b is MSN, MSN-NH₂、MSN-NH₂Cur and MSN-NH₂Zeta potential corresponding to Cur-AOS, c is MSN-NH prepared in different proportions₂Cur absorbance of Cur-AOS nanoparticles.

FIG. 4a shows MSN-NH₂Scanning electron microscope, b is MSN-NH₂AOS scanning electron microscope, c is MSN-NH₂Scanning of-Cur-AOSAn electron microscope; d is MSN-NH₂Transmission electron micrograph of (D), e is MSN-NH₂Transmission electron microscope of-AOS, f is MSN-NH₂Transmission electron micrograph of Cur-AOS.

FIG. 5 is N₂Adsorption-desorption diagram, in which a is MSN and b is MSN-NH₂C is MSN-NH₂-Cur-AOS。

FIG. 6 is a Dynamic Light Scattering (DLS) diagram, where a is MSN and b is MSN-NH₂AOS and c are MSN-NH₂Cur-AOS, d is the thermogravimetric analysis chart of the nanoparticle.

FIG. 7 is an infrared spectrum of the nanoparticle.

FIG. 8 is an XPS spectrum, a is the MSN full spectrum, b is MSN-NH₂Full spectrum, c is MSN-NH₂-AOS full spectrum, d is MSN N1s spectrum, e is MSN-NH₂N1s map, f is MSN-NH₂AOS N1s map.

FIG. 9a shows the solid nuclear magnetic hydrogen spectrum of the nanoparticle, and b shows the structure of the nanoparticle.

FIG. 10 shows the release rate of the Cur nanoparticles affected by different pH values within 24 h; a is MSN-NH₂-Cur nanoparticles, b is MSN-NH₂-Cur-AOS。

FIG. 11 shows Cur, MSN-NH₂-AOS and MSN-NH₂HCT-116 cell viability after 24h treatment with Cur-AOS nanoparticles (. about.p.. about.0.01,. about.p.. about.0.05, the rest not significant).

Detailed Description

The following examples are intended to further illustrate the present invention, but they are not intended to limit or restrict the scope of the invention.

Infrared Spectroscopy (FTIR)

The invention uses infrared spectrum to measure each functional group and analyze the composition of the material. Fully mixing the sample with KBr to prepare a sample, wherein the infrared spectrum scanning range is 4000 to 400cm^-1In between, the instrument resolution is set to 4cm^-1The total number of scans performed was 32.

Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)

The surface morphology of the nanoparticles was evaluated using SEM (S-4800). All nanoparticles were blown evenly onto the silicon wafer, before analysis, a thin gold film was first plated in vacuum, and then the sample was observed at a suitable magnification under an accelerating voltage of 20kV to obtain a clear and regular particle image. TEM images were obtained by Lorentz transmission electron microscopy testing. An appropriate amount of the nanoparticle powder sample was uniformly spread on a conductive copper mesh and TEM scanned.

Dynamic Light Scattering (DLS)

The invention adopts Dynamic Light Scattering (DLS) to detect the whole particle size distribution of the surface of the nano drug-loaded material. Weighing a proper amount of sample to be dissolved in ultrapure water, placing the sample in an ultrasonic wave to realize high dispersion and remove bubbles, and sucking the sample solution to place the sample solution in a sample adaptation pool for analysis.

Thermogravimetric analysis (TGA)

The invention adopts Thermal Gravimetric Analysis (TGA) to quantitatively characterize the specific proportion of the components which can be thermally decomposed and can not be thermally decomposed in the system, and obtains the thermal decomposition temperature of the decomposable part by integrating the TGA curve and the DTA curve. TGA on a model Q50 thermal Analyzer from TA of USA₂The heating was carried out at a temperature rising rate of 10 ℃/min under the atmosphere. Weighing a proper amount of sample, placing the sample in a crucible, and setting a test interval of 37-800 ℃ after the instrument is calibrated. Zeta potential (Zeta-potential) and particle Size (Size) analysis

The invention adopts a Zeta potentiometer to analyze the surface electricity and the particle size distribution of the nanoparticles. Taking a small amount of sample, dispersing with deionized water, performing ultrasonic treatment for 15min before measurement, transferring a proper amount of turbid liquid into a special sample pool, performing measurement in a mode of selecting a dispersant as water, performing three-time parallel test, and taking an average value.

Nitrogen adsorption desorption analysis

The method adopts nitrogen adsorption and desorption to represent the specific surface area, pore volume and pore diameter of the mesoporous material, takes a proper amount of sample to degas and transfers the sample to the liquid nitrogen environment for adsorption-desorption analysis.

X-ray photoelectron spectroscopy (XPS)

The invention utilizes X-ray photoelectron spectroscopy (XPS) to analyze the surface components, and adopts an X-ray photoelectron spectrometer of Kratos company in British to analyze the surface chemical composition of the nano particles and the chemical state of each component in detail.

Nuclear magnetic H-spectrum analysis

The invention adopts nuclear magnetic resonance H spectrum to analyze the material structure. Under the action of an external magnetic field, the energy-level transition generated by the absorption energy of atomic nucleus can form an absorption spectrum, and the chemical structure is deduced by analyzing the chemical shift of H element. Loading Rate (EE) and envelope Rate (LE)

The Encapsulation Efficiency (EE) and the Loading Efficiency (LE) of the nanoparticles are important indexes for judging the quality of the nano-drug, and represent the effects of encapsulation and loading respectively: the mass of Cur initially added is recorded as W_{General assembly}. Centrifuging the nanoparticle sample (8000r/min, 15min) to collect supernatant, W_{Swimming device}Represents the free Cur content, W, in the supernatant_CarrierIs the dry mass of the precipitate. The nanoparticle supernatant prepared in the same way without added Cur was used as a reference, and the unloaded Cur content was determined by UV method according to the standard curve equation at 426 nm. The present invention calculates EE and LE of a drug according to the following formulas (2-1) and (2-2):

in vitro release kinetics experiments

Weighing 5mg of MSN-NH₂-Cur-AOS and MSN-NH₂Cur was added to a centrifuge tube containing 20mL of PBS (pH 7.4 and pH 5.0, containing 4mL of absolute ethanol for curcumin dissolution), and 2 centrifuge tubes were placed in a 37 ℃ constant temperature water bath shaker (150r/min) for in vitro release kinetics of nanoparticles. After 1h, 2h, 4h, 6h, 8h, 12h and 24h, 1mL of the supernatant (supplemented with 1mL of the original pH PBS solution) was centrifuged and returned to the shaker for further shaking. The curcumin concentration is analyzed and detected by an ultraviolet spectrophotometer at 426nm, and the curcumin concentration in the solution is calculated by a standard curve equation.

Preparation example 1 preparation of Mesoporous Silica (MSN)

Dissolving cetyl trimethyl ammonium chloride CTAC (2g) in 20mL of deionized water, dropwise adding 0.32mL of triethanolamine TEA, stirring at 95 ℃ for 1 hour, slowly adding tetraethyl orthosilicate TEOS (1.5mL) into the mixed solution, and reacting for 1 hour; and washing the product with deionized water and ethanol for three times, then carrying out vacuum drying at-60 ℃, and then calcining in a muffle furnace at 550 ℃ for 6 hours to remove the template, thereby obtaining the product, namely Mesoporous Silica (MSN).

Comparative preparation example 1 preparation of Mesoporous Silica (MSN)

0.2g of cetyltrimethylammonium bromide was dissolved in 96mL of water, heated, and stirred. When the temperature reached 80 ℃, 0.7mL of 2M NaOH solution was added to the solution and stirring was continued for 30 minutes. Then 1.4mL of ethyl orthosilicate was slowly added dropwise to the solution, the mixture was stirred vigorously at 80 ℃ for 2 hours, cooled for a while and centrifuged to obtain a white precipitate. Then, the template was removed with 10% ethanol (v/v) hydrochloride solution at reflux for 6h to give the product MSN-1.

Comparative preparation example 2 preparation of Mesoporous Silica (MSN)

CTAB (500mg) was dissolved in a solution containing purified water (200mL), ethylene glycol (40mL) and 1M NaOH (3.5 mL). The mixed solution was heated to 80 ℃ and stirred vigorously for 1 hour. Thereafter, tetraethylorthosilicate TEOS (2.5mL) was added quickly to the above solution and the mixture was held at 80 ℃ for 2h until a white precipitate formed. Centrifuging at 10000 r/m for 20 min, washing the white precipitate with water and ethanol, vacuum drying the white precipitate, and calcining at 550 deg.C for 6 hr to obtain product MSN-2.

TEM scanning of the nanoparticles, Zeta potential (Zeta-potential) and particle Size (Size) analysis

The results of TEM scan, Zeta potential (Zeta-potential) and particle Size (Size) analysis of the MSN-1, MSN-2 and MSN nanoparticles prepared in comparative preparation example 1, comparative preparation example 2 and preparation examples are shown in FIG. 2. in FIG. 2a, no porous structure is observed in the prepared MSN-1, the surface is rough, the particle Size is different, the agglomeration is serious, and the dispersibility is poor. The MSN-2 particles produced in FIG. 2b had a slightly larger particle size compared to MSN-1. As shown in fig. 2c, we can clearly observe that the prepared MSN is a regular spherical particle, the surface is porous, the size is uniform, the particle size is about 50nm, the surface is smooth, and the dispersibility is better than that of the MSN-2 particle. The change of the particle size, the potential and PDI in the modification process is further tested by a Zeta-potentiometer, the difference of the particle sizes of MSN-1, MSN-2 and MSN is obvious in figure 2d, wherein the particle size of the MSN is the minimum, and the surface potentials of three MSN nano particles are-28.3 +/-0.5 mV, -29.6 +/-0.3 mV and-32.2 +/-0.6 mV respectively. PDI also demonstrated that the redispersibility of MSN particles was better than that of particles prepared by the other two methods, which was consistent with TEM map results, and therefore the study selected MSN for subsequent experiments.

Preparation example 2 preparation of aminated MSN nanoparticles

1g of MSN from preparation 1 was reacted with 20mL of 3-aminopropyltriethoxysilane in 100mL of toluene (24h, 60 ℃ C.). After the reaction is finished, the material is washed alternately by ethanol and water to obtain a product MSN-NH₂。

Preparation example 3MSN-NH₂AOS ratio to MSN-NH₂Effect of-Cur-AOS nanoparticles

Under a certain pH condition, MSN-NH₂In solution in the form of cations and AOS in the form of anions, whereby mechanical agitation will cause MSN-NH₂After being uniformly mixed with AOS, the two are self-organized into nano particles through electrostatic interaction. MSN-NH₂The solution at low concentration is clear and transparent, and has almost no flocculation phenomenon, but the solution also contains high-concentration MSN-NH₂Aggregation occurs and a large amount of precipitation occurs. Therefore, experiments need to investigate MSN-NH₂The effect of the ratio to AOS on the morphology of the nanoparticles. As can be seen from FIG. 3a, when MSN-NH is in solution₂When the mass ratio of the MSN to the AOS is 5:1, the MSN-NH is added₂The ratio is relatively high, and a large amount of free MSN-NH still exists at the time₂The obtained nanoparticles have large particle size (320.02 +/-2.30 nm) and poor dispersibility. The reason for this may be that with MSN-NH₂The increase in concentration disrupts the dispersion stability of the particles to cause aggregation of the particles, and ultimately leads to an increase in the particle size of the nanoparticles. With MSN-NH₂Reduction of content (5:1 adjusted to 1:1) the particle size of the nano-particles is reduced (320.02 +/-2.30 nm-236.80 +/-0.54 nm), and the MSN-NH is₂The particles are bound together with the AOS due to positive and negative charge attraction and reach a dispersed steady state. However, when the ratio of the two was adjusted from 1:1 to 1:3, the particle size did not decrease but increased (236.80. + -. 0.54 increased to 269.50. + -. 1.03nm), indicating that a large amount of AOS remained in the nanodispersion at 1: 3. Simultaneously measuring the surface potential of the nano particles along with MSN-NH₂The decrease in the content continued to decrease and was negative, indicating that the potential effect of AOS on the nanoparticles was dominant. In addition, the Zeta potential is in the preparation of MSN-NH₂Changes also occur in the-Cur-AOS process. Preparation of MSN-NH upon functionalization with amine groups, as shown in FIG. 3b₂Thereafter, the zeta potential of MSN increased from-28.6. + -. 1.3mV to 25.3. + -. 0.8 mV. Changes in Zeta potential also indicate MSN-NH₂The synthesis of (2) was successful. MSN-NH after loading curcumin and AOS coating₂The Zeta potential of the anode is respectively converted from 25.3 +/-0.8 mv to 15.3 +/-0.9 mv and-33.1 +/-1.1 mv. The change in zeta potential at each step indicates successful functionalization of the MSN. Fig. 3c shows the absorbance of curcumin released from nanocarriers after loading 0.5mg curcumin, and we can clearly see that the loading rate of curcumin increases with increasing AOS content, and the increase in AOS from 1:1 to 1:3 decreases, and we chose the 1:1 ratio for the subsequent experiments.

Example 1 preparation of alginate-derived oligosaccharide-coated aminated mesoporous silica nanoparticles

Curcumin (Cur)20mg is precisely weighed and added with 20mL of absolute ethyl alcohol to prepare 1mg/mL curcumin solution. Adding 1mL curcumin solution into 10mL Phosphate Buffer Solution (PBS) (pH 7.4: 0.0065mol sodium dihydrogen phosphate (NaH)₂PO₄) And 0.0428mol of disodium hydrogenphosphate (Na)₂HPO₄) To 1 liter of water) and 20mg of MSN-NH₂Stirring at room temperature in dark place for 12 hours; weighing 50mg brown Alginate Oligosaccharide (AOS) powder, dissolving in 50mL ultrapure water to obtain 1% brown alginate oligosaccharide solution, taking 20mL brown alginate oligosaccharide solution, adding 50mg NHS and 0.1g 1-ethyl-carbodiimide (EDC), stirring in dark for 4 hr to activate carboxyl, and adding 10mL MSN-NH after loading with medicine₂The solution was added dropwise to 20mL of activated alginate-derived oligosaccharide solution, and the mixture was placed in a chamberStirring at room temperature in the dark for 12 hours, and centrifuging (8000r/min, 10min) to obtain alginate oligosaccharide coated aminated mesoporous silica nanoparticles MSN-NH₂Cur-AOS, FIG. 1 MSN-NH₂Schematic diagram of preparation of-Cur-AOS nanoparticles. And detecting the absorbance at 426nm by using an ultraviolet spectrophotometer, calculating the concentration of the loaded curcumin through a standard curve, and calculating the drug loading rate of the MSN.

Comparative example 1 MSN-NH not loaded with curcumin₂-AOS nanoparticles

The same method is adopted to prepare MSN-NH without loading curcumin₂-AOS nanoparticles: 20mg of MSN-NH was taken₂Adding 10mL of Phosphate Buffer Solution (PBS), and stirring at room temperature in a dark place for 12 hours; weighing 50mg brown Algae Oligosaccharide (AOS) powder, dissolving in 50mL ultrapure water to obtain 1% brown algae oligosaccharide solution, adding 50mg NHS and 0.1g 1-ethyl-carbodiimide (EDC) into 20mL brown algae oligosaccharide solution, stirring in dark for 4 hr to activate carboxyl, and adding 10mLMSN-NH₂The solution is dripped into 20mL of activated brown algae oligosaccharide solution, stirred at room temperature in the dark for 12 hours and then centrifuged (8000r/min, 10min) to obtain the brown algae oligosaccharide coated aminated mesoporous silica nano particle MSN-NH₂-AOS。

Example 2 SEM and TEM experiments

The blank MSN-NH is shown in the SEM image in FIG. 4a₂Generally, the spherical structure has honeycomb-shaped mesopores. At the same time, the particles begin to aggregate after the polysaccharide AOS is coated as shown in FIG. 4b, which is mainly the binding effect of the linear polysaccharide. After Cur is carried on, MSN-NH₂the-Cur-AOS nanoparticles are significantly larger as shown in FIG. 4 c. After the MSN is subjected to surface functionalization by APTES, loaded with curcumin and gradually conjugated with AOS, TEM images show that the obtained amino-functionalized MSN nanoparticles have the average particle size of about 50nm and a loose mesoporous structure. AOS was observed around the nanoparticles as shown by transmission electron microscopy in FIGS. 4d, 4e and 4f ((d) MSN-NH2, (e) MSN-NH2-AOS and (f) MSN-NH 2-Cur-AOS). Meanwhile, the nano particle size can be clearly seen to be obviously increased, the porosity is gradually reduced, the honeycomb arrangement cannot be clearly observed, and the changes of the shapes can be used as another evidence for the coating of the AOS on the surface of the MSN.

Example 3 analysis of specific surface area of nanoparticles

N₂The adsorption and desorption parameters are shown in table 1 and fig. 5. The experiment employed the BET method to determine MSN (prepared in preparation example 1), MSN-NH₂(prepared in preparation example 2) and MSN-NH₂Cur-AOS (example 4) specific surface areas of 946.6, 648.1 and 286.3m, respectively²The reduction in specific surface area, per gram, indicates that AOS has been successfully coated onto the MSN nanoparticle surface. To a certain extent, the specific surface area can reflect the smoothness of the nanoparticle surface. From the viewpoint of specific surface area, MSN-NH₂The surface of the-Cur-AOS is the roughest, which is essentially consistent with SEM characterization results. The pore size and pore volume of the nanoparticles were analyzed by the BJH method. The pore size was slightly reduced from 5.731 to 4.646nm after ATPES modification, indicating that a small portion of the pores entered the interior of the MSN. After AOS coating, the pore diameter of the sample is obviously reduced (1.126nm), which proves that the alginate oligosaccharide successfully coats the mesoporous silica. The reduction of the specific surface area and the pore size indicates that the alginate oligosaccharides can effectively block the mesoporous pore channels of the MSN, which can help effectively entrap and store the drugs and avoid the leakage and premature release of the drugs.

Table 1 corresponding N₂Adsorption and desorption parameters

Table3-3 The corresponding parameters of N₂ adsorption and desorption.

Example 4 Dynamic Light Scattering (DLS) and thermogravimetric analysis (TGA)

MSN, MSN-NH measurements using Dynamic Light Scattering (DLS) analysis₂-AOS and MSN-NH₂The size of the Cur-AOS nanoparticles. Because the DLS test does not obtain the real particle size of the sample, but the hydraulic diameter of the sample particles in the aqueous solution, the molecules on the surfaces of the nanoparticles can slow down the diffusion speed, and therefore, the data measured by an electron microscope is larger. FIG. 6a shows that the mean size of MSN is approximately 156nm, and after wrapping the alginate oligosaccharide AOS, MSN-NH₂Increase of the AOS mean particle diameter to 306nm (FIG. 6 b). Comparing with fig. 6c, it can be observed that the particle size of the nano-carrier is not greatly affected by the penetration of curcumin into the MSN pores, MSN-NH₂The average particle diameter of the-Cur-AOS particles increased to 313 nm. MSN-NH in contrast to MSN nanoparticles₂-AOS and MSN-NH₂the-Cur-AOS nanoparticles have larger average size and wider size distribution, and the particle sizes of the two are not significantly different. The results prove that the curcumin successfully enters the mesopores of the nano-carrier.

FIG. 6d shows MSN, MSN-NH₂And MSN-NH₂-thermogravimetric analysis (TGA) profile of AOS nanoparticles. As can be seen from the figure, the weight of the three types of nanoparticles is constant to be more than 80% in the temperature range of 30-200 ℃. Within the test range of 30-800 ℃, the loss of pure MSN weight is only 11.1%, and basically before 100 ℃, the loss is mainly bound water on the surface of the MSN nanoparticles, which is probably because the MSN is a compact mesoporous structure. MSN-NH₂The mass loss exists in the temperature ranges of 20-100 ℃ and 400-630 ℃, which is the weight loss phenomenon generated by APTES thermal decomposition and a large amount of hydrothermal volatilization in combination of grafting, and the success of grafting is proved. Furthermore, MSN-NH₂The weight loss of AOS at 20-100 ℃ is caused by surface water detachment, and the weight is greatly reduced at 200-400 ℃, which is related to the thermal degradation of the outer-coated alginate oligosaccharide AOS, and is the result of the oxidative decomposition of the AOS framework, including the degradation of organic framework structure and functional group. The weight loss in the range of 400 to 630 ℃ also indicates NH₂The graft modification was successful with a total weight loss of 62.5%, and these results indicate successful modification of each step.

Example 5 Infrared Spectroscopy

FIG. 7 shows Cur and MSN-NH₂、MSN-NH₂-AOS and MSN-NH₂-infrared spectrum of Cur-AOS nanoparticles. MSN-NH₂、MSN-NH₂-AOS and MSN-NH₂Characteristic peaks of MSN were observed in both-Cur-AOS, 826, 961 and 1084cm^-1The absorption peaks at (A) are the three main characteristic absorption peaks of MSN. The peaks appearing at 826 and 1084 are attributed to the symmetric and antisymmetric stretching vibrations of the Si-O-Si structure in mesoporous silica, respectively. Bending vibration of Si-OH in the nanoparticles formed 961cm^-1The absorption peak at (c). 1405cm^-1And 1256cm^-1Are all composed of-CH ═ CH₂Absorption peaks caused by vibrations. 1645 and 3449cm^-1The peak belongs to H-OH or-OH groups on the surface of the nano particles, which indicates that the surface of the mesoporous silica has a large amount of hydroxyl groups. After binding to AOS, the two peaks broaden and become more pronounced because AOS contains abundant carboxyl groups. The infrared spectrum of the natural curcumin Cur presents a plurality of characteristic bands due to the existence of different functional groups. 3449cm^-1The band at (b) corresponds to a hydroxyl group, which is related to the tensile vibration of the phenolic hydroxyl group. 1424. 1281 and 1155cm^-1The bands at (b) correspond to the stretching vibrations of some of the aromatic and inter-ring chains of the ketone. MSN-NH₂No characteristic functional group absorption peak of curcumin is observed on the Cur-AOS, which also proves that the drug is successfully loaded into the mesoporous structure of the nano carrier.

Example 6X-ray photoelectron spectroscopy (XPS)

X-ray photoelectron spectroscopy (XPS) can determine the bonding energy of sample photoelectrons to infer the composition and structure of sample surface elements. We used XPS to treat MSN, MSN-NH₂And MSN-NH₂AOS nanoparticles were analyzed. As shown in fig. 8a, MSN is simple in structure and contains only Si and O. The Si2p peak and Si2s peak in FIGS. 2-8b and 8C are weakened in intensity, and characteristic peaks of C and N elements appear, which are mainly related to the surface of the C and N elements coated by polysaccharide, and the C and N are mainly derived from APTES and brown algae oligosaccharide, so that the specific gravity of the Si element is reduced. The absence of the N1S peak in MSN is shown in fig. 8 d. FIG. 8e and FIGS. 2-8f are MSN-NH₂And MSN-NH₂The N1s spectrum of AOS. FIG. 8e includes two peaks-NH₂The peak at (401.55eV) and the peak at N- (400.37 eV), mainly from the grafted amino group. As shown in FIG. 8f, AOS wrapped MSN-NH₂A new peak-NH appears₃ ⁺(402.95eV) indicates that the modified amino group can cause the carboxyl group that causes AOS to adsorb to the particle surface through hydrogen bonding and electrostatic attraction. Furthermore, -NH₂The binding energy of the peak and the N-peak is shifted. The results show that the AOS was successfully coated onto the surface of the nanoparticles.

Example 7 Nuclear magnetic H Spectroscopy

By usingNuclear magnetic H-Spectroscopy further used to analyze particle structure, FIG. 9a for MSN and MSN-NH₂Of AOS¹H-NMR spectra, FIGS. 2-9b for MSN and MSN-NH₂-structural diagram of AOS. Wherein, the curve a is MSN, and the curve b is MSN-NH₂-AOS. As can be seen from the curve a, the peak at δ of 3.8ppm is a proton peak in MSN surface bound water. From the curve b, the peak at δ 1.2ppm belongs to-CH on APTES₂-CH₂-formation, the signal peak at δ -2.08 ppm is formed by-O-CO-CH-in AOS, δ -3.2 ppm is-N-CH-after grafting APTES₂The peak of the signal at δ ═ 5.1ppm formed belonged to-HC-O-in AOS.

EXAMPLE 8 drug Release test

To provide sustained drug delivery, a suitable drug delivery system not only exhibits suitable drug loading efficiency and drug loading, but also releases the drug in a controlled manner. The pH of the normal human internal environment is 7.4, and the tumor lesion part is in a slightly acid state, so that the release performance of the drug-loaded nanoparticles under different pH values is investigated. Research shows that the release of the nano drug-loaded particles shows a pH dependence phenomenon, namely the lower the pH value, the higher the release rate. From the results in FIG. 10a, it can be seen that MSN-NH was present in a neutral environment₂The accumulated release amount of the drug in 24 hours of the Cur group only reaches about 41.1 percent, while the release amount in the case of weak acid is about 84.6 percent, which is probably supposed to be that the pH value is reduced, so that the acting force between the drug and the carrier is weakened, and simultaneously, the curcumin in the pores of the aminated mesoporous silica is greatly diffused outwards under the influence of the concentration difference between the inside and the outside of the nano particles, so that the curcumin content in the solution is increased. Shown in FIG. 10b is MSN-NH₂The release curves of curcumin in different pH buffers of Cur-AOS are that in the buffer with pH 7.4, the release speed of curcumin is slowed down, the cumulative release degree in the first two hours is about 12%, the total release amount in 24 hours is 28.9%, compared with MSN-NH₂Cur particles, which show better sustained release effect, due to the coating effect of the brown alga oligosaccharides. At a pH of 5.0, the release rate rose to 67.5%. On one hand, the pH change reduces the dissociation degree of the alginate oligosaccharide, reduces the intermolecular electrostatic interaction, and finally the AOS layer is separated from MSN-NH₂Ginger, shedding of particlesThe flavins were free to diffuse out of the bare MSN lumenal pores into the PBS solution. On the other hand, the concentration difference of the drug inside and outside the nano particles also positively improves the drug release speed. The results show that the brown alga oligosaccharide modification effect can prevent leakage of curcumin in MSN particle pore canals to a certain extent in a neutral environment, the transport of particles in a human body is protected, and the inhibition effect on curcumin in an acidic environment is weakened, so that the good slow release effect of the brown alga oligosaccharide is just proved.

EXAMPLE 9 Loading test of drugs

The loading rate (LE) and the encapsulation rate (EE) are important indexes for judging the quality of the nano material, so that the MSN-NH is considered₂-Cur-AOS and MSN-NH₂Loading performance of Cur systems (drug loading 0.25, 0.50, 0.75 and 1.00 mg). The rich pores of the mesoporous silica nano particles show excellent drug loading effect. The loading LE% of the two groups in table 2 increased with increasing curcumin dose, where the mass of the vehicle used was unchanged, while increasing curcumin dose tended to enter the lumen at the difference in concentration between the inside and outside of the vehicle. The percent EE of encapsulation rate is continuously reduced after the dosage of the curcumin is increased because excessive curcumin exceeds the encapsulation capacity of the nanoparticles. It is the presence of the outer shell of AOS that makes MSN-NH₂The loading effect of the-Cur-AOS group is better than that of MSN-NH₂-a Cur carrier.

TABLE 2 Loading and Release of nanoparticles

Table 2loading and release of nanoparticles

Example 10 cytotoxicity assay

Study the cytotoxicity of the material was evaluated using HCT-116 cells. The study showed that HCT-116 cells were transfected with Cur, MSN-NH₂-AOS and MSN-NH₂Cell viability after 24h treatment with Cur-AOS three groups was dose-related to nanoparticle concentration, and the results are shown in FIG. 11. As can be seen from fig. 11, the rate of decrease in cell survival rate was slow when curcumin alone was contained. Curcumin concentrationWhen 50.0. mu.g/mL is reached, the tumor cells still survive for half, and the cell survival rate is 62.76%. The main reason is that free curcumin cannot rely on endocytosis of cells and can only permeate into the cells, so that the killing effect is weakened. When the concentration is within 50.0 mug/mL, the blank carrier MSN-NH without carrying the medicine₂The AOS has the effect on tumors, the survival rate of cells exceeds 80 percent, the apoptosis is not obvious, and the survival rate is obviously higher than that of cells with the effect of curcumin and drug-loaded nanoparticles at the same concentration. This is just to prove MSN-NH₂AOS vectors are not cytotoxic and are safer. The blank vector at this time obstructs tumor growth and respiration, and is the main cause of apoptosis. To these samples, MSN-NH was added₂The cell survival rate of the experimental group of-Cur-AOS is far lower than that of the other two groups, and the difference is obvious. When the concentration is added to reach 50.0 mug/mL, the cell survival rate is reduced to 27.48%, which shows that the larger the curcumin composite particle dosage is, the higher the probability of cell death is. The results show that the injury effect on the tumor cells is mainly derived from MSN-NH₂Cur-AOS released curcumin, not nanocarriers, and MSN-NH₂AOS is suitable as a carrier for curcumin drugs per se, without causing cytotoxic damage to tumor cells.

The invention provides a core-shell type nano particle, which is prepared by firstly synthesizing aminated mesoporous silica (MSN-NH) in an experiment₂) Then, the brown algae oligosaccharide is functionally modified to a nucleus MSN-NH with positive electricity₂To obtain MSN-NH₂AOS core-shell nanoparticles, whose performance in loading and transporting curcumin was studied. The research adopts chemical characterization methods such as Dynamic Light Scattering (DLS), thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS) and the like to carry out the research on the MSN-NH₂AOS nanoparticles were analyzed. Research optimizes MSN-NH₂The preparation process of the AOS nano-particles and the observation of the loading rate and the encapsulation rate of the nano-particles on curcumin and the in vitro release behavior. The results show that MSN-NH₂The loading rate of the-Cur-AOS nano particles on curcumin reaches 99.46%, the total release amount is 28.9% under a neutral condition, and the total release amount under an acidic environment is 67.5%. Experiment MSN-NH was investigated using MTT and cell uptake experiments₂Cur-AOS nanoparticles have an in vitro anti-tumor effect. Experiment knotThe results show that the MSN-NH produced is comparable to curcumin in the free state₂the-Cur-AOS is more easily taken up by tumor cells, and when the curcumin concentration reaches 50 mu g/mL, the MSN-NH is added₂the-Cur-AOS nano particle has stronger cytotoxicity to tumor cells.

Claims

1. The preparation method of the brown algae oligosaccharide modified aminated mesoporous silica nanoparticle is characterized by comprising the following steps:

1) preparation of mesoporous silica MSN

2) preparation of aminated MSN nanoparticles

Preparing 1mg/mL curcumin solution with anhydrous ethanol, adding phosphoric acid buffer solution and MSN-NH into curcumin solution₂Stirring at room temperature in dark place for 12 hours; obtaining the MSN-NH loaded with curcumin Cur₂A solution;

preparing 1mg/mL brown algae oligosaccharide solution by using ultrapure water, taking the brown algae oligosaccharide solution, adding N-hydroxysuccinimide NHS and 1-ethyl-carbodiimide EDC, stirring for 4 hours in a dark place to activate carboxyl, and obtaining the activated brown algae oligosaccharide solution;

loading curcumin Cur in MSN-NH₂The solution is dripped into the activated brown algae oligosaccharide solution, stirred at room temperature in the dark for 12 hours, and centrifuged at 8000r/min for 10min to obtain the brown algae oligosaccharide coated aminated mesoporous silica nano particle MSN-NH₂-Cur-AOS。

2. The preparation method of the alginate oligosaccharide modified aminated mesoporous silica nanoparticle of claim 1, wherein the weight/volume ratio g/mL of cetyltrimethylammonium chloride to deionized water in step 1) is 1: 10.

3. The preparation method of the alginate oligosaccharide modified aminated mesoporous silica nanoparticle of claim 1, wherein the weight/volume ratio g/mL of cetyltrimethylammonium chloride to triethanolamine in step 1) is 25: 4.

4. The preparation method of the alginate oligosaccharide modified aminated mesoporous silica nanoparticle of claim 1, wherein the weight-to-volume ratio g/mL of cetyltrimethylammonium chloride to tetraethoxysilane in step 1) is 4: 3.

5. The preparation method of the alginate oligosaccharide modified aminated mesoporous silica nanoparticle of claim 1, wherein the weight-to-volume ratio g/mL of MSN to 3-aminopropyltriethoxysilane in the step 2) is 1: 20; the volume ratio of the 3-aminopropyltriethoxysilane to the toluene in the step 2) is 1: 5.

6. The preparation method of the alginate oligosaccharide modified aminated mesoporous silica nanoparticle of claim 1, wherein the pH value of the phosphate buffer solution in the step 3) is 7.4.

7. The preparation method of the alginate oligosaccharide modified aminated mesoporous silica nanoparticle of claim 1, wherein the volume ratio of the curcumin solution to the phosphate buffer solution in the step 3) is 1:10, said MSN-NH₂The weight-to-volume ratio of the curcumin solution is 20: 1.

8. The preparation method of the alginate oligosaccharide modified aminated mesoporous silica nanoparticle of claim 1, wherein the weight/volume ratio of NHS to alginate oligosaccharide solution in step 3) is 5: 2.

9. The preparation method of the alginate oligosaccharide modified aminated mesoporous silica nanoparticle of claim 1, wherein the weight ratio of NHS to 1-ethyl-carbonyldiimine in step 3) is 1: 2.

10. The preparation method of the alginate oligosaccharide modified aminated mesoporous silica nanoparticles of claim 1, wherein the step 3) of loading curcumin with MSN-NH is performed₂The volume ratio of the solution to the activated alginate-derived oligosaccharide solution is 1: 1.