CN113768903B

CN113768903B - Alginate oligosaccharide modified aminated mesoporous silica nanoparticle

Info

Publication number: CN113768903B
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: 2022-12-13
Anticipated expiration: 2041-09-10
Also published as: CN113768903A

Abstract

The invention provides a preparation method of alginate oligosaccharide modified aminated mesoporous silica nanoparticles ₂ ) Then, the brown algae oligosaccharide is functionally modified to a positively charged inner core MSN-NH ₂ To obtain MSN-NH ₂ AOS core-shell nanoparticles, whose performance in loading and transporting curcumin was studied. MSN-NH of the invention ₂ the-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 tools.

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

Mixing MSN with3-aminopropyl triethoxysilane reacts in toluene at the temperature of 60 ℃ for 24 hours, and after the reaction is finished, the material is washed alternately by ethanol and water to obtain a product MSN-NH ₂ ；

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 solution ₂ Stirring at room temperature in dark place for 12 hours; obtaining the MSN-NH loaded with curcumin (Cur) ₂ A 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-NH ₂ The 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-NH ₂ -Cur-AOS。

Preferably, the weight-to-volume ratio g/mL of the hexadecyltrimethylammonium chloride to the deionized water in the step 1) is 1.

Preferably, the weight-to-volume ratio g/mL of the hexadecyltrimethylammonium chloride to the triethanolamine in the step 1) is 25.

Preferably, the weight-to-volume ratio g/mL of the hexadecyltrimethylammonium 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; the volume ratio of the 3-aminopropyltriethoxysilane to the toluene in 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 phosphate buffer solution in the step 3) is 1:10, said MSN-NH ₂ The weight-to-volume ratio of mg/ml to curcumin solution was 20.

Preferably, the weight-to-volume ratio mg/ml of the NHS to the alginate-derived oligosaccharide solution in 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) ₂ The 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 invention ₂ the-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 MSN-1,2 and MSN-2,3 respectively.

FIG. 3a shows MSN-NH prepared in different ratios ₂ The particle size and zeta potential of AOS, b is MSN, MSN-NH ₂ 、MSN-NH ₂ Cur and MSN-NH ₂ Zeta potential corresponding to Cur-AOS, c is MSN-NH prepared in different proportions ₂ Cur absorbance of Cur-AOS nanoparticles.

FIG. 4a shows MSN-NH ₂ Scanning electron microscope, b is MSN-NH ₂ AOS scanning electron microscope, c is MSN-NH ₂ -scanning electron microscopy of Cur-AOS; d is MSN-NH ₂ Transmission electron micrograph of (D), e is MSN-NH ₂ Transmission electron microscopy of AOS, f is MSN-NH ₂ Transmission electron micrograph of Cur-AOS.

FIG. 5 is N ₂ Adsorption-desorption diagram, in which a is MSN and b is MSN-NH ₂ C is MSN-NH ₂ -Cur-AOS。

FIG. 6 is a Dynamic Light Scattering (DLS) diagram, where a is MSN and b is MSN-NH ₂ AOS and c are MSN-NH ₂ Cur-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-NH ₂ Full spectrum, c is MSN-NH ₂ -AOS full spectrum, d MSN 1s spectrum, e MSN-NH ₂ N1s map, f is MSN-NH ₂ AOS N1s map.

Fig. 9a is the solid nuclear magnetic hydrogen spectrum of the nanoparticle, and b is 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-NH ₂ Cur nanoparticles, b is MSN-NH ₂ -Cur-AOS。

FIG. 11 shows Cur, MSN-NH ₂ -AOS and MSN-NH ₂ HCT-116 cell survival after 24h treatment with Cur-AOS nanoparticles (indicated by P < 0.01, indicated by P < 0.05, not significant otherwise).

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 ^-1 In between, the instrument resolution is set to 4cm ^-1 The 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 thermogravimetric 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 DTA curve thereof to characterize the thermal decomposition temperature of the decomposable part by integrating the TGA curve. TGA on a model Q50 thermal Analyzer from TA in USA ₂ The 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 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 invention adopts nitrogen gas adsorption and desorption to represent the specific surface area, pore volume and pore diameter of the mesoporous material, and takes a proper amount of sample to be degassed and then 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, energy absorption energy of atomic nucleus generates energy level transition to form an absorption spectrum, and the chemical structure is presumed 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 (8000 r/min,15 min) to collect supernatant, W _{Swimming device} Represents the free Cur content, W, in the supernatant _Carrier Is 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 EE and LE of the drug are calculated according to the following formulas (2-1) and (2-2):

in vitro release kinetics experiments

Weighing 5mg of MSN-NH ₂ -Cur-AOS and MSN-NH ₂ Cur 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 (150 r/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 (2 g) in 20mL of deionized water, dropwise adding 0.32mL of triethanolamine TEA, stirring at 95 ℃ for 1 hour, slowly adding tetraethyl orthosilicate TEOS (1.5 mL) 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 ℃,0.7 ml of 2M NaOH solution was added to the solution and stirring was continued for 30 minutes. 1.4mL of ethyl orthosilicate was then slowly added dropwise to the solution, the mixture was stirred vigorously at 80 ℃ for 2 hours, cooled for a period of time and centrifuged to give 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 (500 mg) was dissolved in a solution containing purified water (200 mL), ethylene glycol (40 mL) and 1M NaOH (3.5 mL). The mixed solution was heated to 80 ℃ and stirred vigorously for 1 hour. Thereafter, tetraethylorthosilicate TEOS (2.5 mL) 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, zeta-potential (Zeta-potential) and Size (Size) analysis of the nanoparticles

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, it can be clearly observed that the prepared MSN is regular spherical particles, has a porous surface, is uniform in size, has a particle size of about 50nm, has a smooth surface, and has better dispersibility than MSN-2 particles. 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 are 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-NH ₂ 。

Preparation example 3MSN-NH ₂ AOS ratio to MSN-NH ₂ Effect of-Cur-AOS nanoparticles

At a certain pH, MSN-NH ₂ In solution in the form of cations and AOS in the form of anions, whereby mechanical agitation will mix MSN-NH ₂ After being uniformly mixed with AOS, the two are self-organized into nano particles through electrostatic interaction. MSN-NH ₂ The solution at low concentration is clear and transparent, and has almost no flocculation phenomenon, but the solution also contains high-concentration MSN-NH ₂ Aggregation occurs and a large amount of precipitation occurs. Therefore, experiments need to investigate MSN-NH ₂ The effect of the ratio to AOS on the morphology of the nanoparticles. As can be seen from FIG. 3a, when MSN-NH is in solution ₂ The mass ratio of the MSN to the AOS is 5:1, due to MSN-NH ₂ The ratio is relatively high, and a large amount of free MSN-NH still exists at the time ₂ The obtained nanoparticles have large particle size (320.02 +/-2.30 nm) and poor dispersibility. The reason for this may be that with MSN-NH ₂ The 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-NH ₂ The content is reduced (5:1 is adjusted to 1:1), the particle size of the nano particles is reduced (320.02 +/-2.30 nm to 236.80 +/-0.54 nm), and the MSN-NH is reduced ₂ The 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 these two was adjusted from 1:1 to 1:3, the particle size did not decrease or increase (236.80. + -.0.54 increased to 269.50. + -.1.03 nm), indicating that 1:3 contained a large amount of AOS in the nanodispersions. Measure simultaneouslyThe surface potential of the obtained nano particles is dependent on MSN-NH ₂ The 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-NH ₂ Changes also occur in the-Cur-AOS process. Preparation of MSN-NH upon functionalization with amine groups, as shown in FIG. 3b ₂ Thereafter, the zeta potential of MSN increased from-28.6. + -. 1.3mV to 25.3. + -. 0.8mV. Changes in Zeta potential also indicate MSN-NH ₂ The synthesis of (2) was successful. MSN-NH after loading curcumin and AOS coating ₂ The 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 by nanocarriers after loading 0.5mg curcumin, we can clearly see that as the content of AOS increases, the loading rate of curcumin increases and the AOS increases from 1:1 to 1:3, and considering all, we have selected 1:1 for the subsequent experiment.

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) ₂ PO ₄ ) And 0.0428mol of disodium hydrogenphosphate (Na) ₂ HPO ₄ ) To 1 liter of water) and 20mg of MSN-NH ₂ Stirring 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 medicine ₂ The solution is dripped into 20mL of activated brown algae oligosaccharide solution, stirred at room temperature in the dark for 12 hours and then centrifuged (8000 r/min,10 min) to obtain the brown algae oligosaccharide coated aminated mesoporous silica nano particle MSN-NH ₂ Cur-AOS, FIG. 1 MSN-NH ₂ Schematic diagram of preparation of-Cur-AOS nanoparticles. Detecting absorbance at 426nm with ultraviolet spectrophotometer, and calculating curcumin concentration by standard curveAnd (4) calculating the drug loading rate of the MSN.

Comparative example 1 MSN-NH not loaded with curcumin ₂ -AOS nanoparticles

The same method is adopted to prepare MSN-NH without loading curcumin ₂ -AOS nanoparticles: 20mg of MSN-NH was taken ₂ Adding 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-NH ₂ The solution is dripped into 20mL of activated brown algae oligosaccharide solution, stirred at room temperature in the dark for 12 hours and then centrifuged (8000 r/min,10 min) to obtain the brown algae oligosaccharide coated aminated mesoporous silica nano particle MSN-NH ₂ -AOS。

Example 2 SEM and TEM experiments

The blank MSN-NH is shown in the SEM image in FIG. 4a ₂ Generally, 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 loaded, MSN-NH ₂ the-Cur-AOS nanoparticles are significantly larger as shown in FIG. 4c. 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 can be observed around the nanoparticles as in transmission electron microscopy 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

N ₂ The 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-NH ₂ (prepared according to preparation example 2) and MSN-NH ₂ Cur-AOS (example 4) specific surface areas of946.6 648.1 and 286.3m ² The reduction in specific surface area, g, 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 aspect of specific surface area, MSN-NH ₂ The surface of the-Cur-AOS is the roughest, which is substantially consistent with SEM characterization results. The pore size and pore volume of the nanoparticles were analyzed by BJH method. The pore diameter is slightly reduced from 5.731 to 4.646nm after ATPES modification, which indicates that a small part enters the interior of the MSN. After AOS coating, the pore diameter of the sample is obviously reduced (1.126 nm), 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 pores of the MSN, which can help to effectively entrap and store the drug and avoid the leakage and premature release of the drug.

Table 1 corresponding N ₂ Adsorption and desorption parameters

Table3-3 The corresponding parameters of N ₂ adsorption and desorption.

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

MSN, MSN-NH were measured using Dynamic Light Scattering (DLS) analysis ₂ -AOS and MSN-NH ₂ The 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 average size of MSN is about 156nm, and after AOS encapsulation, MSN-NH ₂ The AOS average particle size increased 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-NH ₂ The average particle diameter of the-Cur-AOS particles increased to 313nm. MSN-NH in contrast to MSN nanoparticles ₂ -AOS and MSN-NH ₂ Cur-AOS nanoparticles have a larger average size and a broader size distribution, and ofThe particle size difference is not significant. The results prove that the curcumin successfully enters the mesopores of the nano-carrier.

FIG. 6d shows MSN, MSN-NH ₂ And MSN-NH ₂ -thermogravimetric analysis (TGA) profile of AOS nanoparticles. As can be seen from the figure, the weight of the three types of nanoparticles was constant at 80% or more in the temperature range of 30 to 200 ℃. The loss of pure MSN weight was only 11.1% in the 30-800 ℃ test range, and essentially the bound water on the surface of the MSN nanoparticles was lost before 100 ℃, probably because MSN is a dense mesoporous structure. MSN-NH ₂ The mass loss exists in the temperature ranges of 20-100 ℃ and 400-630 ℃, which is the weight loss phenomenon generated by the thermal decomposition and a large amount of combined hydrothermal volatilization of the grafted organic substance APTES, and the success of grafting is proved. Furthermore, MSN-NH ₂ The weight loss of AOS at 20-100 ℃ is caused by surface water detachment, and at 200-400 ℃ the weight is greatly reduced, which is related to the thermal degradation of the outer coating alginate oligosaccharide AOS, which is the result of the oxidative decomposition of the AOS backbone, including the degradation of organic backbone structure and functional groups. The weight loss in the range from 400 to 630 ℃ is likewise stated for NH ₂ The 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-NH ₂ 、MSN-NH ₂ -AOS and MSN-NH ₂ -infrared spectrum of Cur-AOS nanoparticles. MSN-NH ₂ 、MSN-NH ₂ -AOS and MSN-NH ₂ Characteristic peaks of MSN were observed in both-Cur-AOS, 826, 961 and 1084cm ^-1 The 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 ^-1 The absorption peak at (c). 1405cm of ^-1 And 1256cm ^-1 Are all made of-CH ═ CH ₂ Absorption peaks caused by vibrations. 1645 and 3449cm ^-1 The 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 broadened and were more pronounced becauseAOS is rich in carboxyl groups. The natural curcumin Cur presents a plurality of characteristic bands in the infrared spectrum due to the existence of different functional groups. 3449cm ^-1 The band at (b) corresponds to a hydroxyl group, which is related to the tensile vibration of the phenolic hydroxyl group. 1424. 1281 and 1155cm ^-1 The bands at (b) correspond to the stretching vibration of some aromatic rings and inter-ring chains of the ketone. MSN-NH ₂ No 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-NH ₂ And MSN-NH ₂ AOS 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, and the characteristic peaks of C and N elements appear, which are mainly related to the surface of the Si element is coated by polysaccharide, and the C and N are mainly derived from APTES and alginate oligosaccharide, so that the specific gravity of the Si element is reduced. No N1S peak in MSN is shown in fig. 8 d. FIG. 8e and FIGS. 2-8f are MSN-NH ₂ And MSN-NH ₂ -N1 s spectrum of AOS. FIG. 8e includes two peaks-NH ₂ Peak at (401.55 eV) and = N- (400.37 eV), mainly from grafted amino groups. As shown in FIG. 8f, AOS wrapped MSN-NH ₂ A new peak-NH appears ₃ ⁺ (402.95 eV) indicates that the modified amino groups can cause the carboxyl groups of the AOS to adsorb to the particle surface through hydrogen bonding and electrostatic attraction. Furthermore, -NH ₂ Peak and = N-peak binding energy shifts. The results show that the AOS was successfully coated onto the surface of the nanoparticles.

Example 7 Nuclear magnetic H Spectroscopy

Further analysis of the particle structure by NMR spectroscopy, FIG. 9a shows MSN and MSN-NH ₂ Of AOS ¹ H-NMR spectra, FIGS. 2-9b for MSN and MSN-NH ₂ -structural drawing of AOS. Wherein, the curve a is MSN, and the curve b is MSN-NH ₂ -AOS. As can be seen from curve a, the signal peak at δ =3.8ppm belongs to the proton peak in MSN surface bound water. From curve bIt can be seen that the signal peak at δ =1.2ppm belongs to-CH on APTES ₂ -CH ₂ Formation, the signal peak at δ =2.08ppm is formed by-O-CO-CH-in AOS, δ =3.2ppm is-N-CH-after grafting APTES ₂ The signal peak formed at δ =5.1ppm belongs 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 of FIG. 10a, it can be seen that MSN-NH is present in a neutral environment ₂ The 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-NH ₂ The 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-NH ₂ Cur 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 alginate oligosaccharide, reduces the intermolecular electrostatic interaction, and finally the AOS layer is separated from MSN-NH ₂ The particles fell off the surface and curcumin was free to dissociate from the bare MSN lumen pores and diffuse 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 algae oligosaccharide modification effect can prevent curcumin in MSN particle pore canals from leaking to a certain extent in a neutral environment and protect the particles in a human bodyWhile the inhibition effect on curcumin in an acidic environment is weakened, which just proves the good slow-release effect of the brown alga oligosaccharide.

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 considered ₂ -Cur-AOS and MSN-NH ₂ Loading performance of the Cur system (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-NH ₂ The loading effect of the group-Cur-AOS is better than that of MSN-NH ₂ -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-NH ₂ -AOS and MSN-NH ₂ Cell 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. When the curcumin concentration reaches 50.0 mu g/mL, 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 medicine ₂ AOS on tumors, cell survival of more than 80%, cellsThe apoptosis is not obvious, and the survival rate is obviously higher than that of the cells acted by the curcumin and the drug-loaded nanoparticles with the same concentration. This is just to prove MSN-NH ₂ AOS 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 added ₂ The 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 main source of the injury effect on the tumor cells is MSN-NH ₂ Cur-AOS released curcumin, not nanocarriers, and MSN-NH ₂ AOS 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 experiment ₂ ) Then, the brown algae oligosaccharide is functionally modified to a nucleus MSN-NH with positive electricity ₂ To obtain MSN-NH ₂ AOS 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-NH ₂ AOS nanoparticles were analyzed. Research optimizes MSN-NH ₂ The preparation process of the AOS nano-particles, and the loading rate and the encapsulation rate of the AOS nano-particles on curcumin and in-vitro release behavior are examined. The results show that MSN-NH ₂ The loading rate of the-Cur-AOS nano particles to 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 experiments ₂ In vitro anti-tumor effect of Cur-AOS nanoparticles. The experimental result shows that compared with curcumin in a free state, the prepared MSN-NH is ₂ the-Cur-AOS is more easily taken up by tumor cells, and when the curcumin concentration reaches 50 mu g/mL, the MSN-NH is added ₂ the-Cur-AOS nano particle has strong 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

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, respectively washing products with the 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 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 to obtain product MSN-NH ₂ ；

3) Preparation of alginate oligosaccharide coated aminated mesoporous silica nano particle

Preparing 1mg/mL curcumin solution with anhydrous ethanol, adding phosphoric acid buffer solution and MSN-NH into curcumin solution ₂ Stirring at room temperature in dark place for 12 hours; obtaining the MSN-NH loaded with curcumin Cur ₂ A 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 a dark place to activate carboxyl to obtain the activated brown algae oligosaccharide solution;

loading curcumin Cur in MSN-NH ₂ The 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-NH ₂ -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.

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

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

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

7. The preparation method of the alginate oligosaccharide modified aminated mesoporous silica nanoparticles 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-NH ₂ The weight-to-volume ratio of the curcumin solution is 20.

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 performed ₂ The volume ratio of the solution to the activated alginate-derived oligosaccharide solution is 1:1.