Preparation method and application of an oral drug-carrying dual-drive nanomotor

Abstract

The invention relates to a preparation method and application of an oral drug-carrying dual-drive nanomotor, which effectively solves the preparation problem of the oral drug-carrying dual-drive nanomotor and realizes the application problem in the preparation of anti-colorectal cancer drugs. Mesoporous polydopamine nanoparticles and compound BNN6 are prepared, and a mesoporous polydopamine nanoparticle solution and a BNN6 solution are stirred by π-π conjugation to obtain PDA-BNN6; mesoporous silica nanospheres are prepared, MSN is adsorbed on liquid paraffin, and stirred with (3-mercaptopropyl)trimethoxysilane for thiolization, and PDA-BNN6 is modified to one side of the MSN surface to obtain an MSN / PB dual-sphere nanomotor; CP and MSN / PB are added to an N,N-dimethylformamide solution, and magnetic stirring is performed to obtain CP@MSN / PB, a Lactobacillus rhamnosus bacterial liquid is taken, and its cell wall is extracted, and the cell wall is mixed with the CP@MSN / PB, and extruded to obtain an oral drug-carrying dual-drive nanomotor CP@MSN / PB@CWL. The product preparation method of the present invention is scientific and advanced, the product performance is good, oral administration, the ability to penetrate the intestinal barrier of colorectal cancer is strong, the drug utilization rate is high, the efficacy is good, and the operation is simple and convenient.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a preparation method and application of an oral drug-carrying dual-drive nanomotor. Background Art

[0002] At present, surgery combined with chemotherapy is the main treatment for advanced colorectal cancer in the clinic, but chemotherapy has problems such as high toxicity. Oral nano drug delivery systems can reduce drug toxicity, improve efficacy and are safer and more convenient. However, the dense and thick intestinal mucus layer and the tightly connected intestinal epithelial barrier are limited in the oral nano drug delivery process, making it difficult for drugs to reach effective therapeutic concentrations. The autonomous movement of nano motors provides technical support for drugs to cross intestinal mucus. Among them, near-infrared (NIR) laser-propelled nano motors have broad application prospects in penetrating complex physiological barriers due to their characteristics of no fuel and stable motion performance. However, for crossing the complex depth of physiological barriers in the body, the penetration ability of NIR is limited, and other additional driving sources are needed to extend the penetration depth and motion time of nano motors. The current method for nano drug delivery systems to cross the intestinal epithelial barrier is mainly through targeting receptors on intestinal epithelial cells and endocytosis mediated by transporters. However, if the substances that enter the intestinal epithelial cells cannot be quickly excreted through transcytosis, they may be re-excreted into the intestinal lumen by biological structures such as P-glycoprotein on the basolateral side of the intestinal epithelial cells, or rapidly degraded by intracellular lysosomes, which poses great difficulties to the intestinal epithelial cell transcytosis pathway of oral drug delivery systems.

[0003] The inflammatory environment of colorectal cancer secretes a large amount of IgA immunoglobulin. The glycan structure rich in the cell wall of Lactobacillus rhamnosus (Cell Wellof LGG, CWL) can specifically bind to IgA immunoglobulin and is resistant to gastric acid digestion. Therefore, it can specifically recognize IgA immunoglobulin and achieve efficient colonization of colorectal cancer in the intestine. In order to autonomously and efficiently penetrate intestinal mucus, mesoporous SiO2 nanoparticles (MSN, mesoporous silica nanoparticles) with high drug loading were used as the drug-carrying element of the chemotherapy drug CP, and mesoporous polydopamine (PDA) with good photothermal performance and safety was used as the propulsion element. The NO donor N,N-di-sec-butyl-N,N-dinitro-1,4-phenylenediamine (BNN6) was combined with PDA through π-π conjugation (PDA-BNN6). A nanomaterial was designed using Pickering emulsion, in which the MSN part was exposed for chemical adsorption of the PDA part through thiol bonds, so that a gas- and near-infrared dual-driven autonomous motion nanomotor (MSN / PB) could be constructed. At the same time, endothelial relaxing factor (NO) can also enhance the permeability of the intestinal epithelial barrier by directly affecting free radical activity and cell signaling, thereby downregulating the expression of intestinal epithelial cell tight junction proteins such as ZO-1, ZO-3 and occludens, and achieving efficient penetration of the intestinal epithelial barrier. After reaching the colon, the bacterial cell wall will degrade in the colon microenvironment, releasing the drug-loaded nanomotors to kill tumor cells, thereby improving the drug delivery efficiency and the efficiency of colorectal cancer treatment. However, there has been no public report on how to prepare oral drug-loaded dual-drive nanomotors and use them to prepare anti-colorectal cancer drugs. Summary of the invention

[0004] In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a method for preparing an oral drug-loaded dual-drive nanomotor and its application, which can effectively solve the problem of preparing oral drug-loaded dual-drive nanomotor and realize its application in the preparation of anti-colorectal cancer drugs.

[0005] The technical solution of the present invention is a method for preparing an oral drug-loaded dual-drive nanomotor, comprising the following steps:

[0006] 1) Preparation of PDA-BNN6 nanoparticles:

[0007] A. In a 50 ml mixed solution of ethanol and water in a volume ratio of 1:1, add 1.0 g triblock copolymer Pluronic F127 (referred to as F127) and 0.4 g dopamine hydrochloride (DA), stir at room temperature, and after mixing evenly, add 1,3,5-trimethylbenzene (TMB) dropwise, the mass ratio of F127 to TMB is 1:1-2, stir at room temperature to form a white emulsion, then slowly add 5 mL of ammonia water, stir at 50 ° C for 1 hour, and then centrifuge at 10000 rpm for 15 minutes, collect the solid precipitate, and then ultrasonically wash the solid precipitate 3 times with a mixed solution of 20 ml ethanol and 10 ml acetone each time, each washing for 30 minutes, to obtain mesoporous polydopamine nanoparticles (PDA);

[0008] B. Dissolve 2.34 mL of N,N-di-sec-butyl-1,4-phenylenediamine (BPA) in 18 mL of ethanol, add 20 mL of 6 M degassed sodium nitrite (NaNO2), stir for 30 min under nitrogen protection, add 20 mL of 6 M concentrated hydrochloric acid solution dropwise with a separatory funnel, stir for 4 h, centrifuge at 10000 rpm for 30 min, collect the solid precipitate, wash the solid precipitate with 10 ml of water and 10 ml of ethanol solution three times, each washing for 10 min, to obtain compound BNN6;

[0009] C. Add 2 mg of mesoporous polydopamine nanoparticles (PDA) prepared in A to 4 mL of DMSO solution under constant stirring, dissolve the compound BNN6 prepared in B in DMSO solution to a concentration of 1 mg / mL, stir 4 ml of PDA solution and 2 ml of BNN6 solution at room temperature for 12 h through π-π conjugation, centrifuge at 12000 rpm for 15 min, collect the solid precipitate, and wash it three times with excess water, each time for 10 min, to obtain PDA-BNN6, PDA and BNN6, according to the mass ratio of 1:1 to 2;

[0010] 2) Preparation of MSN / PB double-ball nanomotors:

[0011] Add 68 mg of triethanolamine (TEA) to 25 mL of water, stir gently with magnetic force at 80 ° C for 30 min, then add 380 mg of hexadecyltrimethylammonium bromide (CTAB) and 40 mg of sodium salicylate (NaSal) as structure directing agents, continue stirring for 1 h, then add 4 mL of tetraethoxysilane (TEOS), stir for 12 h, centrifuge at 12000 rpm for 20 min to collect the solid product, wash it 3 times with 20 ml of anhydrous ethanol, 10 min each time, remove the residual reactants, and then reflux with 1 M concentrated HCL methanol solution at 60 ° C for 6 h, a total of 3 times, to remove the template CTAB and NaSal to form mesoporous silica nanospheres;

[0012] MSN was adsorbed on liquid paraffin, wherein the mass of liquid paraffin was 1 g, and the mass ratio of liquid paraffin to MSN was 100:1-2. The mixture was homogenized by a homogenizer for 10 min, and reacted with magnetic stirring at 75°C for 1 h to form a Pickering emulsion; cooled to room temperature, diluted with 10 mL of methanol, and stirred with 200 μL of (3-mercaptopropyl)trimethoxysilane at room temperature for 3 h to perform thiolization;

[0013] Subsequently, the prepared PDA-BNN6 was modified to one side of the MSN surface. The mass ratio of PDA-BNN6 and MSN was 1-2:1. The mixture was stirred in Tris-HCl (trishydroxymethylaminomethane hydrochloride) pH8.5 buffer at room temperature overnight for 8-12 hours. The paraffin was washed with chloroform to prepare the MSN / PB double-ball nanomotor. The dosage ratio of PDA-BNN6, MSN, and Tris-HCl pH8.5 buffer was 1:1:1.

[0014] 3) Preparation of oral drug-loaded dual-drive nanomotors CP@MSN / PB@CWL:

[0015] CP and MSN / PB were added to 10 mL N,N-dimethylformamide (DMF) solution, the mass ratio of drug CP to nanoparticle MSN / PB was 1:1-2, the drug CP was 10 mg, magnetic stirring was performed at room temperature for 12 h, centrifuged at 12000 rpm for 15 min, the solid precipitate was collected, and the solid precipitate was washed with 10 ml of physiological saline for 10 min to obtain CP@MSN / PB;

[0016] 10 mL of Lactobacillus rhamnosus bacterial solution was taken and centrifuged at 4°C and 6000 rpm for 10 min to obtain a precipitate, which was then washed twice with 10 ml pH 7.4 PBS, each time for 10 min. 1 mL of lysozyme at a concentration of 1 mg / mL was added and incubated in a 37°C water bath for 20 min to obtain a bacterial solution. The bacterial solution was taken out and added with 10 mL pH 7.4 PBS and dispersed in a 100 kDa ultrafiltration tube, centrifuged at 4°C and 5000 rpm for 10 min, and 15 ml pH 7.4 PBS was added to wash twice, each time for 15 min to remove the unbroken Escherichia coli and bacterial contents to obtain the Lactobacillus rhamnosus biofilm, which was resuspended in 10 mL PBS and its cell wall (CWL) was extracted. 10 ml CWL was evenly mixed with CP@MSN / PB at a mass ratio of 1:1-3, and extruded by direct extrusion to obtain the oral drug-carrying dual-drive nanomotor CP@MSN / PB@CWL.

[0017] Application of the oral drug-loaded dual-drive nanomotor CP@MSN / PB@CW prepared by the method of the present invention in the preparation of anti-colorectal cancer drugs.

[0018] The product preparation method of the present invention is scientific and advanced, the product performance is good, oral administration, the ability to penetrate the intestinal barrier of colorectal cancer is strong, the drug utilization rate is high, the efficacy is good, the operation is simple and convenient, it has broad application prospects, and huge economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the TEM result of PDA of the present invention.

[0020] Figure 2 This is the TEM result of the MSN of the present invention.

[0021] Figure 3 This is the TEM result of MSN / PDA of the present invention.

[0022] Figure 4 This is the CLSM result diagram of the co-localization of the bacterial film and MSN / PDA of the present invention.

[0023] Figure 5 The graph is a curve showing the release results of NO in simulated colon fluid of the present invention.

[0024] Figure 6 This is a graph showing the MSD results of the MSN / PB@CWL of the present invention.

[0025] Figure 7 These are the results of the effects of the nanocarriers of the present invention on the viability of Caco2 cells.

[0026] Figure 8 These are the results of the effects of the nanocarriers of the present invention on the viability of CT26 cells. Implementation

[0027] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0028] The present invention can be specifically implemented by the following examples. Example

[0029] The present invention provides a method for preparing an oral drug-carrying dual-drive nanomotor, comprising the following steps:

[0030] 1) Preparation of PDA-BNN6 nanoparticles:

[0031] A. In a mixed solution of 25 mL ethanol and 25 mL water, add 1.0 g triblock copolymer Pluronic F127 (referred to as F127) and 0.4 g dopamine hydrochloride (DA), stir at room temperature, and after mixing evenly, add 1.6 mL of 1,3,5-trimethylbenzene (TMB) dropwise, stir at room temperature to form a white emulsion, then slowly add 5 mL of ammonia water, stir at 50 ° C for 1 h, and then centrifuge at 10000 rpm for 15 min, collect the solid precipitate, and then ultrasonically wash the solid precipitate with 20 ml ethanol and 10 ml acetone for 3 times, each washing for 30 min, to obtain mesoporous polydopamine nanoparticles (PDA);

[0032] B. Dissolve 2.34 mL of N,N-di-sec-butyl-1,4-phenylenediamine (BPA) in 18 mL of ethanol, add 20 mL of 6 M degassed sodium nitrite (NaNO2), stir for 30 min under nitrogen protection, add 20 mL of 6 M concentrated hydrochloric acid solution dropwise with a separatory funnel, stir for 4 h, centrifuge at 10000 rpm for 30 min, collect the solid precipitate, wash the solid precipitate with 10 ml of water and 10 ml of ethanol solution three times in sequence, each washing for 10 min, to obtain compound BNN6;

[0033] C. Add 2 mg of mesoporous polydopamine nanoparticles (PDA) prepared in A to 4 mL of DMSO solution under constant stirring, and add 2 mL of DMSO solution of compound BNN6 with a concentration of 1 mg / mL dropwise, stir at room temperature for 12 h through π-π conjugation, centrifuge at 12000 rpm for 15 min, collect the solid precipitate, and then wash it three times with excess water, each washing for 10 min, to obtain PDA-BNN6;

[0034] 2) Preparation of MSN / PB double-ball nanomotors:

[0035] 68 mg of triethanolamine (TEA) was added to 25 mL of water, and the mixture was gently stirred magnetically at 80 °C for 30 min. Then, 380 mg of hexadecyltrimethylammonium bromide (CTAB) and 40 mg of sodium salicylate (NaSal) were added as structure directing agents, and the stirring was continued for 1 h. Then, 4 mL of tetraethoxysilane (TEOS) was added, and the mixture was stirred for 12 h. The solid product was collected by centrifugation at 12000 rpm for 20 min, and the solid product was washed with 20 ml of anhydrous ethanol for 3 times, each time for 10 min, to remove the residual reactants, and then refluxed with 1 M concentrated HCL methanol solution at 60 °C for 6 h, for a total of 3 times, to wash away the template CTAB and NaSal to form mesoporous silica nanospheres.

[0036] 20 mg of MSN was adsorbed on 1 g of liquid paraffin, and the mixture was homogenized at 15000 rpm for 10 min by a homogenizer, and then reacted with magnetic stirring at 75°C for 1 h to form a Pickering emulsion; cooled to room temperature, diluted with 10 mL of methanol, and stirred with 200 μL of (3-mercaptopropyl)trimethoxysilane at room temperature for 3 h to perform thiolation;

[0037] Subsequently, the prepared PDA-BNN6 was modified to one side of the MSN surface. The mass ratio of PDA-BNN6 and MSN was 1:1. The mixture was stirred in Tris-HCl (trishydroxymethylaminomethane hydrochloride) pH 8.5 buffer at room temperature overnight for 8-12 hours. The paraffin was washed with chloroform to prepare the MSN / PB double-ball nanomotor. The dosage ratio of PDA-BNN6, MSN, and Tris-HCl buffer was 1:1:1.

[0038] 3) Preparation of oral drug-loaded dual-drive nanomotors CP@MSN / PB@CWL:

[0039] 10 mg of CP and 10 mg of MSN / PB were added to 5 mL of N,N-dimethylformamide (DMF) solution, magnetically stirred at room temperature for 12 h, centrifuged at 12000 rpm for 15 min, and the solid precipitate was collected. The solid precipitate was washed with 10 ml of physiological saline for 10 min to obtain CP@MSN / PB;

[0040] Take 10mL of Lactobacillus rhamnosus bacterial solution, centrifuge at 4℃ and 6000rpm for 10min to obtain a precipitate, wash the precipitate twice with 10mlpH7.4 PBS, add 1mL of 1mg / mL lysozyme, and incubate in a 37℃ water bath for 20min to obtain a bacterial solution; take out the bacterial solution, add 10mL pH7.4 PBS and disperse it in a 100kDa ultrafiltration tube, centrifuge at 4℃ and 5000rpm for 10min, add 15ml pH7.4 PBS and wash twice, each time for 15min, remove the unbroken Escherichia coli and bacterial contents, and obtain the Lactobacillus rhamnosus biofilm, resuspend it in 10mL PBS, squeeze it through 800, 400, and 220μm filter membranes to extract its cell wall (CWL); 10ml CWL and CP@MSN / PB were uniformly mixed in a mass ratio of 1:1-3 and extruded by direct extrusion to obtain the oral drug-loaded dual-drive nanomotor CP@MSN / PB@CWL. Example

[0041] The present invention provides a method for preparing an oral drug-loaded dual-drive nanomotor, wherein the mass ratio of the triblock copolymer PluronicF127 and 1,3,5-trimethylbenzene (TMB) is preferably 1:1.5;

[0042] The mass ratio of the mesoporous polydopamine nanoparticles (PDA) and the compound BNN6 is preferably 1:1;

[0043] The mass ratio of cetyltrimethylammonium bromide (CTAB) to sodium salicylate (NaSal) is preferably 9.5:1;

[0044] The mass ratio of the MSN to the liquid paraffin is preferably 50:1;

[0045] The pickering emulsion was stirred at 15000 rpm for 10 min using a homogenizer;.

[0046] The Tris-HCl is 10 mM and has a pH of 8.5. Example

[0047] The present invention discloses a method for preparing an oral drug-loaded dual-drive nanomotor, wherein the loaded drug is cisplatin and the ultrafiltration tube is 100KDa;

[0048] The mass ratio of the biofilm to CP@MSN / PB is preferably 2:1;

[0049] The direct extrusion was performed 5 times with 800, 400, and 220 micron filter membranes respectively;

[0050] The particle size of the oral drug-loaded dual-drive nanomotor is 150-180 nm.

[0051] It should be noted that the above embodiments are only used to illustrate the specific implementation of the technical solution of the present invention, rather than to limit the scope of protection. Although the present invention is described in detail with reference to the preferred embodiments, ordinary technicians in the field should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution. Essentially the same technical solutions should all be covered by the scope of protection of the present invention.

[0052] The oral drug-loaded dual-drive nanomotor CP@MSN / PB@CWL prepared by the method of the present invention has a strong ability to penetrate the intestinal barrier of colorectal cancer, and can be effectively used to prepare anti-colorectal cancer drugs, realize the application of oral drug-loaded dual-drive nanomotor CP@MSN / PB@CWL in the preparation of anti-colorectal cancer drugs, and obtain very good beneficial technical effects through experiments. The relevant experimental data are as follows:

[0053] The present invention uses transmission electron microscopy to analyze the morphology of PDA-BNN6, the scale bar is 100nm, and the results are as follows Figure 1 As shown, from Figure 1 From the analysis, we can see that the average size of mesoporous polydopamine nanoparticles is 80 nm;

[0054] The morphology of the MSN / PB double-ball nanomotor was analyzed using transmission electron microscopy. The scale bar is 100 nm. Figure 2 , 3 shown; from Figure 2 , 3 From the analysis, it can be seen that the average particle size of MSN nanoparticles is 80nm, and the average size of MSN / PB double-sphere nanoparticles is 160nm;

[0055] The particle size analyzer was used to perform fluid dynamics analysis on the potential level and particle size distribution of the oral drug-carrying dual-drive nanomotor CP@MSN / PB@CWL, and the transmission electron microscope was used to analyze its morphology. Figure 4 , the potential of PDA is -22.49mV, the potential of MSN is -6.316mV, and the potential of MSN / PDA is -27.12mV. After CWLs are coated on the surface of MSN / PDA, the potential is similar to that of macrophage membrane, which is 11.02mV, indicating that CWL is successfully covered on the surface of MSN / PDA. At the same time, after the DiI-labeled bacterial film and FITC-labeled MSN / PDA are mixed, there is a certain overlap, which proves that the co-localization of bacterial film and MSN / PDA can be achieved.

[0056] 1) NO release efficiency of CP@MSN / PB@CWL nanomotors

[0057] Specific experimental method: 0.5 mg / ml BNN6 and PDA-BNN6 were mixed in simulated colonic fluid and irradiated for 10 minutes without laser irradiation and under 808 nm laser of 1.0 W cm-2. Subsequently, 50 μL of supernatant obtained by centrifugation was collected for quantitative detection of released NO. The suspension was then placed in the dark for 20 minutes, and 50 μL of supernatant was taken out every 10 minutes for measurement. After reacting with Greiss reagent, the absorption of the collected supernatant was immediately measured by an enzyme reader at a wavelength of 540 nm to detect the release rate of NO in simulated colonic fluid in vitro.

[0058] The results are as follows Figure 5 As shown, NO can only be released when stimulated by near-infrared light, and the release amount can reach 11.2μM in 90 minutes.

[0059] 2) Experiment on the autonomous motion ability of CP@MSN / PB@CWL nanomotors

[0060] Specific experimental methods: The multi-particle tracking (MPT) method was used to analyze the movement of particles in mucus under a confocal microscope. 0.02 mg of PDA-BNN6, MSN / PDA, MSN / PB, and MSN / PB@CWL were added to 1 ml of mucin solution and mixed well. The results were investigated in the presence or absence of NO and 0, 0.5, 1, and 1.5 W cm-2 The motion trajectory of nanoparticles under 808nm near-infrared laser irradiation for 10s was analyzed, and the mean square displacement (MSD) of different nanoparticles was analyzed.

[0061] The result is like Figure 6 As shown in the figure, with the increase of laser power, the mean square displacement (MSD) of MSN / PB@CWL increased significantly, indicating that the oral dual-drive nanomotor has good autonomous movement ability.

[0062] 3) In vitro biosafety study of CP@MSN / PB@CWL nanomotors

[0063] Specific experimental method: CaCO2 and CT26 cells were cultured at 8×10 3 / well were inoculated into 96-well plates, with 6 replicate wells in each group. After 12 h of culture, the plates were washed with PBS three times, each time for 2 min, and 200 μL of drug-containing culture medium was added, which were divided into MSN / PDA, MSN / PB, MSN / PB@CWL, and MSN / PB@CWL+NIR, where NIR was 808 nm; 1.5 W cm -2 ; 10min, drug concentrations were 5, 10, 20, 50, 100, 150, 200μg / ml. After 24h of culture, the drug-containing medium was discarded, and the cells were washed with PBS 3 times, 2min each time. 10μL of CCK8 and 90μL of culture medium were added to each well, mixed, and incubated for another 3h. The absorbance at OD450nm was detected with an enzyme-labeled instrument, and the cell survival rate was calculated to evaluate the in vitro safety.

[0064] The results are as follows Figure 7 , 8 As shown in the figure, when the nanocarriers were incubated with the two cells at different concentrations for 24 h, the cell viability was above 90%. -2 After irradiation for 10 minutes, there was no obvious toxicity, which proved that the nanocarrier had good biosafety and low toxicity.

[0065] Experiments show that the present invention provides a method for preparing an oral drug-loaded dual-drive nanomotor and its application in the preparation of anti-colorectal cancer drugs to solve the problem that drugs are difficult to cross the intestinal barrier. The polysaccharide structure rich in CWL can specifically bind to IgA immunoglobulin and is resistant to gastric acid digestion, thereby achieving efficient colonization of colorectal cancer in the intestinal tract. MSN with a high drug loading is used as the drug loading element of the chemotherapy drug CP, and PDA with good photothermal performance and safety is used as a propulsion element. The NO donor BNN6 is combined with PDA through π-π conjugation (PDA-BNN6). A nanomaterial is designed using Pickering emulsion, in which the MSN part is exposed and used to chemically adsorb the PDA part through a thiol bond, so that a gas and near-infrared dual-driven autonomous motion nanomotor (MSN / PB) can be constructed. At the same time, NO can also enhance the permeability of the intestinal epithelial barrier by directly affecting free radical activity and cell signal transduction, thereby downregulating the expression of intestinal epithelial cell tight junction proteins such as ZO-1, ZO-3 and occludens, and achieving efficient penetration of the intestinal epithelial barrier. After reaching the colon, the bacterial cell wall will be degraded in the colon microenvironment, releasing the drug-loaded nanomotors to kill tumor cells, thereby improving the efficiency of drug delivery and the efficiency of colorectal cancer treatment.

[0066] Compared with the prior art, the present invention has the following beneficial effects:

[0067] 1) The oral drug-carrying dual-drive nanomotor of the present invention has abundant synthetic materials, is easy to obtain, has low cost, and is convenient for clinical transformation;

[0068] 2) The oral drug-carrying dual-drive nanomotor provided by the present invention, with the help of the inflammatory chemotaxis of the bacterial film, can resist the harsh environment of the stomach during oral administration and achieve efficient colonization in the intestinal area of ​​colorectal cancer.

[0069] 3) The oral drug-carrying dual-drive nanomotor provided by the present invention solves the limitation of dense and thick intestinal mucus layer and tightly connected intestinal epithelial barrier in the process of oral nano-drug delivery for the treatment of colorectal cancer. The whole system has the advantages of low toxicity, good water solubility, strong stability, good biocompatibility, and no obvious toxic side effects; it uses near-infrared and NO dual-drive nanomotor to achieve efficient penetration of intestinal mucus and intestinal epithelial barrier at the same time, and finally achieves drug release at the cancer site, with significant anti-tumor activity, which is a major innovation in the treatment of colorectal cancer drugs, has the value of promoting practical applications, and has huge economic and social benefits.

Claims

1. A method for preparing an oral drug-loaded dual-drive nanomotor, characterized in that: The following steps are involved: 1) Preparation of PDA-BNN6 nanoparticles: A. In a 50 ml mixed solution of ethanol and water in a volume ratio of 1:1, add 1.0 g triblock copolymer Pluronic F127 and 0.4 g dopamine hydrochloride (DA), stir at room temperature, and after mixing evenly, add 1,3,5-trimethylbenzene (TMB) dropwise, the mass ratio of F127 to TMB is 1:1-2, stir at room temperature to form a white emulsion, then slowly add 5 mL of ammonia water, stir at 50 ° C for 1 hour, and then centrifuge at 10000 rpm for 15 minutes, collect the solid precipitate, and then ultrasonically wash the solid precipitate 3 times with a mixed solution of 20 ml ethanol and 10 ml acetone, each washing for 30 minutes, to obtain mesoporous polydopamine nanoparticles (PDA); B. Dissolve 2.34 mL of N,N-di-sec-butyl-1,4-phenylenediamine (BPA) in 18 mL of ethanol, add 20 mL of 6 M degassed sodium nitrite (NaNO2), stir for 30 min under nitrogen protection, add 20 mL of 6 M concentrated hydrochloric acid solution dropwise with a separatory funnel, stir for 4 h, centrifuge at 10000 rpm for 30 min, collect the solid precipitate, wash the solid precipitate with 10 ml of water and 10 ml of ethanol solution three times, each washing for 10 min, to obtain compound BNN6; C. Add 2 mg of mesoporous polydopamine nanoparticles (PDA) prepared in A to 4 mL of DMSO solution under constant stirring, dissolve the compound BNN6 prepared in B in DMSO solution to a concentration of 1 mg / mL, stir 4 ml of PDA solution and 2 ml of BNN6 solution at room temperature for 12 h through π-π conjugation, centrifuge at 12000 rpm for 15 min, collect the solid precipitate, and wash it three times with excess water, each time for 10 min, to obtain PDA-BNN6, PDA and BNN6, according to the mass ratio of 1:1 to 2; 2) Preparation of MSN / PB double-ball nanomotors: 68 mg of triethanolamine (TEA) was added to 25 mL of water, and the mixture was gently stirred magnetically at 80 °C for 30 min. Then, 380 mg of hexadecyltrimethylammonium bromide (CTAB) and 40 mg of sodium salicylate (NaSal) were added as structure directing agents, and the stirring was continued for 1 h. Then, 4 mL of tetraethoxysilane (TEOS) was added, and the mixture was stirred for 12 h. The solid product was collected by centrifugation at 12000 rpm for 20 min, and the solid product was washed with 20 ml of anhydrous ethanol for 3 times, each time for 10 min, to remove the residual reactants, and then refluxed with 1 M concentrated HCL methanol solution at 60 °C for 6 h, for a total of 3 times, to remove the template CTAB and NaSal, and to form mesoporous silica nanospheres. MSN was adsorbed on liquid paraffin, wherein the mass of liquid paraffin was 1 g, and the mass ratio of liquid paraffin to MSN was 100:1-2. The mixture was homogenized by a homogenizer for 10 min, and reacted with magnetic stirring at 75°C for 1 h to form a Pickering emulsion; cooled to room temperature, diluted with 10 mL of methanol, and stirred with 200 μL of (3-mercaptopropyl)trimethoxysilane at room temperature for 3 h to perform thiolization; Subsequently, the prepared PDA-BNN6 was modified onto one side of the MSN surface, with a mass ratio of PDA-BNN6 to MSN of 1-2:1, stirred overnight at room temperature for 8-12 hours in Tris-HCl pH8.5 buffer, and the paraffin was washed with chloroform to prepare the MSN / PB double-ball nanomotor; wherein the amount ratio of PDA-BNN6, MSN, and Tris-HCl pH8.5 buffer was 1:1:1; 3) Preparation of oral drug-loaded dual-drive nanomotors CP@MSN / PB@CWL: CP and MSN / PB were added to 10 mL N,N-dimethylformamide (DMF) solution, the mass ratio of drug CP to nanoparticle MSN / PB was 1:1-2, the drug CP was 10 mg, magnetic stirring was performed at room temperature for 12 h, centrifuged at 12000 rpm for 15 min, solid precipitates were collected, and the solid precipitates were washed with 10 ml of physiological saline for 10 min to obtain CP@MSN / PB; the CP was cisplatin; 10 mL of Lactobacillus rhamnosus bacterial solution was taken and centrifuged at 4°C and 6000 rpm for 10 min to obtain a precipitate, which was then washed twice with 10 ml of pH 7.4 PBS, each time for 10 min. 1 mL of lysozyme at a concentration of 1 mg / mL was added and incubated in a 37°C water bath for 20 min to obtain a bacterial solution. The bacterial solution was taken out and added with 10 mL of pH 7.4 PBS and dispersed in a 100 kDa ultrafiltration tube, centrifuged at 4°C and 5000 rpm for 10 min, and 15 ml of pH 7.4 PBS was added to wash twice, each time for 15 min to remove the unbroken Escherichia coli and bacterial contents to obtain a Lactobacillus rhamnosus biofilm, which was resuspended in 10 mL of PBS to extract its cell wall (CWL). 10 ml of CWL was evenly mixed with CP@MSN / PB at a mass ratio of 1:1 to 3, and extruded by direct extrusion to obtain an oral drug-carrying dual-drive nanomotor CP@MSN / PB@CWL.

2. The method for preparing the oral drug-carrying dual-drive nanomotor according to claim 1, characterized in that: The following steps are involved: 1) Preparation of PDA-BNN6 nanoparticles: A. In a mixed solution of 25 mL ethanol and 25 mL water, add 1.0 g triblock copolymer Pluronic F127 and 0.4 g dopamine hydrochloride (DA), stir at room temperature, and after mixing evenly, add 1.6 mL 1,3,5-trimethylbenzene (TMB) dropwise, stir at room temperature to form a white emulsion, then slowly add 5 mL ammonia water, stir at 50 ° C for 1 h, then centrifuge at 10000 rpm for 15 min, collect the solid precipitate, and then ultrasonically wash the solid precipitate with 20 ml ethanol and 10 ml acetone for 3 times, each washing for 30 min, to obtain mesoporous polydopamine nanoparticles (PDA); B. Dissolve 2.34 mL of N,N-di-sec-butyl-1,4-phenylenediamine (BPA) in 18 mL of ethanol, add 20 mL of 6 M degassed sodium nitrite (NaNO2), stir for 30 min under nitrogen protection, add 20 mL of 6 M concentrated hydrochloric acid solution dropwise with a separatory funnel, stir for 4 h, centrifuge at 10000 rpm for 30 min, collect the solid precipitate, wash the solid precipitate with 10 ml of water and 10 ml of ethanol solution three times in sequence, each washing for 10 min, to obtain compound BNN6; C. Add 2 mg of mesoporous polydopamine nanoparticles (PDA) prepared in A to 4 mL of DMSO solution under constant stirring, and add 2 mL of DMSO solution of compound BNN6 with a concentration of 1 mg / mL dropwise, stir at room temperature for 12 h through π-π conjugation, centrifuge at 12000 rpm for 15 min, collect the solid precipitate, and then wash it three times with excess water, each washing for 10 min, to obtain PDA-BNN6; 2) Preparation of MSN / PB double-ball nanomotors: 68 mg of triethanolamine (TEA) was added to 25 mL of water, and the mixture was gently stirred magnetically at 80 °C for 30 min. Then, 380 mg of hexadecyltrimethylammonium bromide (CTAB) and 40 mg of sodium salicylate (NaSal) were added as structure directing agents, and the stirring was continued for 1 h. Then, 4 mL of tetraethoxysilane (TEOS) was added, and the mixture was stirred for 12 h. The solid product was collected by centrifugation at 12000 rpm for 20 min, and the solid product was washed with 20 ml of anhydrous ethanol for 3 times, each time for 10 min, to remove the residual reactants, and then refluxed with 1 M concentrated HCL methanol solution at 60 °C for 6 h, for a total of 3 times, to wash away the template CTAB and NaSal to form mesoporous silica nanospheres. 20 mg of MSN was adsorbed on 1 g of liquid paraffin, and the mixture was homogenized at 15000 rpm for 10 min by a homogenizer, and then reacted with magnetic stirring at 75°C for 1 h to form a Pickering emulsion; cooled to room temperature, diluted with 10 mL of methanol, and stirred with 200 μL of (3-mercaptopropyl)trimethoxysilane at room temperature for 3 h to perform thiolation; Subsequently, the prepared PDA-BNN6 was modified onto one side of the MSN surface, with a mass ratio of PDA-BNN6 to MSN of 1:1, and stirred overnight at room temperature for 8-12 hours in Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride) pH 8.5 buffer, and the paraffin was washed with chloroform to prepare the MSN / PB double-ball nanomotor; wherein the amount ratio of PDA-BNN6, MSN, and Tris-HCl buffer was 1:1:1; 3) Preparation of oral drug-loaded dual-drive nanomotors CP@MSN / PB@CWL: 10 mg of CP and 10 mg of MSN / PB were added to 5 mL of N,N-dimethylformamide (DMF) solution, magnetically stirred at room temperature for 12 h, centrifuged at 12000 rpm for 15 min, and the solid precipitate was collected. The solid precipitate was washed with 10 ml of physiological saline for 10 min to obtain CP@MSN / PB; Take 10mL of Lactobacillus rhamnosus bacterial solution, centrifuge at 4℃ and 6000rpm for 10min to obtain a precipitate, wash the precipitate twice with 10ml pH7.4PBS, add 1mL of lysozyme with a concentration of 1mg / mL, and incubate in a 37℃ water bath for 20min to obtain a bacterial solution; take out the bacterial solution, add 10mL pH7.4 PBS and disperse it in a 100kDa ultrafiltration tube, centrifuge at 4℃ and 5000rpm for 10min, add 15ml pH7.4 PBS and wash twice, each time for 15min, remove the unbroken Escherichia coli and bacterial contents, and obtain the Lactobacillus rhamnosus biofilm, resuspend it in 10mL PBS, squeeze it through 800, 400, and 220μm filter membranes to extract its cell wall (CWL); 10ml CWL and CP@MSN / PB were uniformly mixed in a mass ratio of 1:1-3 and extruded by direct extrusion to obtain the oral drug-loaded dual-drive nanomotor CP@MSN / PB@CWL.

3. The method for preparing the oral drug-loaded dual-drive nanomotor according to claim 1, characterized in that: The mass ratio of the triblock copolymer Pluronic F127 and 1,3,5-trimethylbenzene (TMB) is 1:1.5; The mass ratio of the mesoporous polydopamine nanoparticles (PDA) and the compound BNN6 is 1:1; The mass ratio of the MSN to the liquid paraffin is 50:1; The pickering emulsion was stirred at 15000 rpm for 10 min using a homogenizer; The Tris-HCl is 10 mM and has a pH of 8.

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4. Use of the oral drug-loaded dual-drive nanomotor CP@MSN / PB@CW prepared by the method described in any one of claims 1 to 3 in the preparation of anti-colorectal cancer drugs.

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