CN112870387B

CN112870387B - Magnetic nano-drug carrier and preparation method and application thereof

Info

Publication number: CN112870387B
Application number: CN202110226819.XA
Authority: CN
Inventors: 吴卓; 张汉臣; 邓立; 刘海晴; 麦思瑶
Original assignee: Sun Yat Sen Memorial Hospital Sun Yat Sen University
Current assignee: Sun Yat Sen Memorial Hospital Sun Yat Sen University
Priority date: 2021-02-26
Filing date: 2021-02-26
Publication date: 2023-08-29
Anticipated expiration: 2041-02-26
Also published as: CN112870387A

Abstract

The invention relates to the technical field of drug carriers, in particular to a magnetic nano drug carrier and a preparation method and application thereof. The magnetic nano-drug carrier comprises a coupling product of SP94 targeting peptide and an siRNA delivery carrier, wherein the coupling product of the siRNA delivery carrier comprises superparamagnetic iron oxide nanoparticles, quaternary ammonium cationized amylose and quaternary ammonium cationized amylose-tetraphenyl ethylene-superparamagnetic iron oxide nanoparticles formed by coupling of tetraphenyl ethylene, and the siRNA is adsorbed in the magnetic nano-drug carrier. The cytotoxicity experiment proves that the nano-drug carrier has good biocompatibility, good stability, targeting property and drug release, and can be applied to preparing cancer gene therapy drugs.

Description

Magnetic nano-drug carrier and preparation method and application thereof

Technical Field

The invention relates to the technical field of drug carriers, in particular to a magnetic nano drug carrier and a preparation method and application thereof.

Background

In recent years, the magnetic nano particles are widely applied to fields of biological treatment, diagnosis and the like due to the unique properties, the surface chemical activity of the magnetic nano particles can be easily combined with biological macromolecules, the magnetic nano particles become a good targeting carrier through modifying specific ligands on the surfaces of the magnetic nano particles, the magnetic nano particles can absorb electromagnetic wave energy to be converted into heat energy under the action of an alternating magnetic field, the heat energy can be limited to tumor tissues, and apoptosis and necrosis of cells can be caused when the temperature exceeds 41 ℃, so that the thermal therapy of tumors can be realized.

In clinical treatment, low toxicity and high efficiency of drugs are always important points of research, and research on targeted drug delivery, namely how to realize the effect of high-efficiency drug treatment, is increasingly paid attention to by researchers. Folic acid is a small molecular weight vitamin, has the characteristics of stable structure, low price, no immunogenicity and the like compared with proteins such as a single molecular antibody and the like, has strong binding force between folic acid and a folic acid receptor, can be efficiently mediated into tumor cells, and is a targeting substance with high application value. Based on the property that folic acid receptors are expressed in large amounts in the cell membranes of most malignant tumors, but normal cells have few expressions, studies have been conducted on the linkage of folic acid to drug carriers and nanoparticles made of them through chemical bonds, so that folic acid/folic acid receptor-mediated targeted delivery is a research hotspot in this field.

Magnetic nanoparticle research and development has been focused on particle size, modification of surface molecules, targeting, etc., and magnetic nanoparticles are used in various nanobiotechnology such as molecular imaging using Magnetic Resonance Imaging (MRI), disease tracking and diagnosis, hyperthermia, drug delivery, magnetic biosensors, and microfluidic systems. In particular, the magnetic nanoparticles can be used as diagnostic probes for MRI.

However, the nano-drug carrier reported in the prior literature has the defects of (1) large toxicity, (2) difficult degradation, difficult living body tracing and the like, so that the use of the nano-drug carrier is limited to a certain extent.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a magnetic nano-drug carrier and a preparation method thereof, wherein the SP94 targeting peptide is coupled with an siRNA delivery carrier, the magnetic nano-drug carrier has better safety, stability, transfection efficiency and biocompatibility, and the magnetic nano-drug carrier combines real-time tracing, controllable siRNA release and gene therapy, so that the optimal strategy of anti-tumor treatment with low side effect is realized.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

in a first aspect, the present invention provides a magnetic nano-drug carrier, the magnetic nano-drug carrier comprises a coupling product of an SP94 targeting peptide and an siRNA delivery carrier, the coupling product of the siRNA delivery carrier comprises superparamagnetic iron oxide nanoparticles, quaternary ammonium cationized amylose and quaternary ammonium cationized amylose-tetraphenyl ethylene-superparamagnetic iron oxide nanoparticles formed by coupling tetraphenyl ethylene, and the siRNA is adsorbed in the magnetic nano-drug carrier.

The RNAi technology of the invention improves the safety, stability, biocompatibility and transfection efficiency of the nano drug carrier. The vectors of the present invention have higher efficiency in transportation across cell membranes than other vectors, and can prevent excretion if the size and surface coating of the vector are appropriate; organic nanoparticles including cationic polymer nanoparticles, lipid-based systems, etc., into which the drug may be incorporated by chemical bonding or physical entrapment; cationic polymer nanoparticles have a number of advantages: 1. low toxicity, good biocompatibility and high safety; 2. biodegradable, low immunogenicity; 3. the surface of the nano-carrier is positively charged; 4. high gene transfection efficiency; 5. batch production, low cost; 6. higher thermodynamic and dynamic stability. The amylose-composed nanoparticles used in the present invention have higher biocompatibility in addition to the common advantages of cationic polymer nanoparticles.

The invention utilizes the MRI targeting feature with SPOI to monitor the nano particles in vitro, the SPIO has superparamagnetism, the particles have no magnetism under the condition of no external magnetic field, when placed in the external magnetic field, the crystals of the particles are aligned and generate very high local magnetic field gradient, thereby leading water protons to spin and shift phase, and further reducing the relaxation time of T1WI and T2WI of surrounding water magnetic fields; thus, SPIO produces a low signal with a sequence of T2WI, and can improve image quality and contrast; the SPIO has good drug effect and pharmacokinetics characteristics, wherein the iron has biocompatibility and can be reused or recovered by cells through physiological iron metabolism; the surface of the SPIO is coated with polysaccharide, polyethylene glycol (PEG), polypyrrole (PPy), polylactic acid (PLA) and the like, so that the biocompatibility can be improved, and the SPIO which is a classical T2WI negative contrast agent can be selected for biodegradability, so that the SPIO has high biocompatibility and low toxicity, and has the function of MRI visualization;

The SP94 targeting peptide has high specific affinity to various human hepatocellular carcinoma cell lines, the affinity of the SP94 targeting peptide to the hepatocellular carcinoma cells is 1 ten thousand times higher than that of normal hepatocytes, endothelial cells, peripheral blood mononuclear cells, B lymphocytes and T lymphocytes, and the accurate targeting specificity of the SP94 targeting peptide is combined with a delivery system, so that the targeting property of the SP94 targeting peptide can be enhanced.

After synthesis of the oleylamine and oleic acid stable hydrophobic SPIO, tetraphenyl ethylene (TPE) and SPIO are both hydrophobic in nature, and TPE is introduced into the core of SPIO nanoparticle by way of physical entrapment through hydrophobic interactions. Thus, quaternary ammonium cationized amylose-superparamagnetic iron oxide nanoparticles can incorporate a large amount of hydrophobic TPE and SPIO in the inner core while maintaining water solubility with the hydrophilic outer shell of quaternary ammonium cationized amylose.

The magnetic nano-drug carrier prepared by the invention has good effect of releasing Survivin siRNA, and releases 50.0% of Survivin siRNA after 5 hours and 79.8% after 24 hours.

As a preferred embodiment of the magnetic nano-drug carrier, the particle size of the superparamagnetic iron oxide nano-particles is 5-8 nm.

As a preferred embodiment of the magnetic nano-drug carrier, the particle size of the magnetic nano-drug carrier is 100-200 nm.

As a preferred embodiment of the magnetic nano-drug carrier of the present invention, the surface potential of the magnetic nano-drug carrier is 5.8mV, and the polymer dispersibility index of the magnetic nano-drug carrier is 0.25.

As a preferred embodiment of the magnetic nano-drug carrier of the present invention, the preparation method of the quaternary ammonium cationized amylose comprises the following steps:

adding amylose into distilled water, adjusting pH=12-14 with NaOH solution, heating and stirring, slowly dripping aqueous solution of active etherifying agent, and continuously stirring for reacting for 12 hours; after the reaction is finished, the pH value is regulated to be neutral by hydrochloric acid solution, and the solution is dialyzed, filtered and freeze-dried by a cellulose dialysis bag with the molecular weight cutoff of 8000-14000 Da to obtain the quaternary ammonium cationic amylose.

The framework of the drug carrier adopts amylose, is a natural macromolecule with good biocompatibility and degradability, the loaded MRI contrast agent is superparamagnetic iron oxide nanoparticle (SPIO), and folic acid used for modifying the carrier is a small molecular weight vitamin, and has stable structure and no immunogenicity.

The invention selects safe and low-toxicity substances on the manufacturing materials of the nano-drug carrier, adopts a one-pot method to synthesize, has the advantages of no intermediate separation and purification and simplified operation, and ensures the purity of the synthesized carrier.

The second aspect of the present invention provides a method for preparing the magnetic nano-drug carrier, comprising the following steps:

s1, adding quaternary ammonium cationized amylose into distilled water to dissolve to form quaternary ammonium cationized amylose water solution, and weighing FeCl ₃ ·6H ₂ O and FeCl ₂ ·4H ₂ O is dissolved in distilled water, then the distilled water is added into a quaternary ammonium cationized amylose water solution to form a mixture I, the mixture is heated in a water bath, ammonia water is added, the temperature is reduced after the reaction is finished, the mixture is dialyzed and centrifuged to obtain a supernatant, and the quaternary ammonium cationized amylose-superparamagnetic iron oxide nanoparticle water solution is obtained;

s2, dissolving tetraphenyl ethylene in CH ₂ Cl ₂ Adding the quaternary ammonium cationic amylose-superparamagnetic iron oxide nanoparticle aqueous solution obtained in the step S1, uniformly mixing to form a mixture II, dripping the mixture II into pure water in ultrasound, dialyzing, freeze-drying, and collecting a solid product to obtain the quaternary ammonium cationic amylose-tetraphenylethylene-superparamagnetic iron oxide nanoparticle aqueous solutionIron oxide nanoparticles;

s3, dissolving the SP94 targeting peptide in DMSO, adding EDC, HCl and NHS, stirring in a dark place to obtain a DMSO solution of the SP94 targeting peptide active ester, adding the aqueous solution of the quaternary ammonium cationic amylose-tetraphenyl ethylene-superparamagnetic iron oxide nanoparticles into the DMSO solution of the SP94 targeting peptide active ester in the step S2 to form a mixture III, reacting in a dark place, dialyzing, centrifuging, and taking supernatant to obtain an aqueous solution of the SP94 targeting peptide targeted modified quaternary ammonium cationic amylose-tetraphenyl ethylene-superparamagnetic iron oxide nanoparticles;

S4, diluting the aqueous solution of the SP94 targeting peptide targeting modified quaternary ammonium cationic amylose-tetraphenylethylene-superparamagnetic iron oxide nano-particles in the step S3, adding the siRNA aqueous solution, and mixing to form the magnetic nano-drug carrier.

As the preparation method of the magnetic nano-drug carrier, the concentration of ammonia water in the step S1 is 25% by mass.

As the preparation method of the magnetic nano-drug carrier, the molecular weight of the dialysis interception mixture I in the step S1 is 8000-14000 Da; the molecular weight of the dialysis interception mixture II in the step S2 is 2000Da; the molecular weight of the dialysis interception mixture III in the step S3 is 8000-14000 Da.

As the preparation method of the magnetic nano-drug carrier, in the step S4, the molar ratio of nitrogen in the aqueous solution of the SP94 targeting peptide targeting modified quaternary ammonium cationic amylose-tetraphenylethylene-superparamagnetic iron oxide nanoparticle to phosphorus in the aqueous solution of siRNA is 5:1.

The third aspect of the invention provides application of the magnetic nano-drug carrier in preparing cancer gene therapy drugs.

The invention also utilizes bimodal imaging combining living body fluorescence imaging and magnetic resonance imaging to evaluate the therapeutic effect of the medicine in vitro. Compared with the traditional imaging technology, the fluorescent imaging has the advantages of low cost, high sensitivity, convenient use and the like, can be used for monitoring the spatial-temporal distribution of molecular processes and biological structures in real time, and traditional fluorophores are usually used in a solution at low concentration, because the ACQ effect caused by aggregation is quenched at high concentration, thereby limiting the further application of the fluorophores in ultrasensitive analysis and long-term monitoring; fluorophores with AIE properties emit weakly when dissolved in solution, but exhibit fluorescence in the aggregated state, which function allows them to function at high concentrations, giving them excellent sensitivity and photostability. AIE luminophores with corresponding ligands are particularly suitable for photoimaging cancer cells with a high signal-to-noise ratio, the fluorophores emit little light in aqueous solution but become emitted after internalization into the cancer cells by endocytosis, so that cancer cell imaging can be conveniently carried out in a wash-free manner. MRI is a non-invasive imaging modality, without ionizing radiation, and T2WI MRI sequences can show inflammation and edema; while conventional MRI can only perform qualitative image analysis based on arbitrary unit signal intensity analysis, T2 mapping evaluates relaxation times based on voxel-wise basis to enable non-invasive visualization and quantification of tissue constituents, T2 mapping has the potential to be a "non-invasive" biopsy because it reflects tissue constituents, especially free water content, and is sensitive to tissue hydration or edema, without the need for contrast agents. The invention quantitatively analyzes the T2WI signal change by using a classical T2 mapping method, obtains the signal change similar to a fluorescence image, and proves that the change of the tumor signal influenced by the nano particles can be better analyzed by quantitatively analyzing the MRI image. The multi-mode imaging can be combined with a plurality of imaging means to realize the complementary advantages of each imaging method, the application range of each imaging method is enlarged, and the integrated MRI and optical imaging can provide high resolution, selectivity and sensitivity.

And the invention also injects nano drug carriers with different doses into the tumor-bearing nude mouse model to perform MR scanning and living body fluorescence imaging, analyzes the relation between MR signals and SPIO content, and explores the tumor/organ distribution and the body clearance rate of TPE fluorescent nano particles in living bodies. Selecting a ROI for a tumor, extracting quantitative texture parameters of an image, combining a pathological slide, respectively selecting the whole tumor, an active tumor growth region and a necrotic region, comparing the differences of the ROI texture parameters and the TPE fluorescence intensities of different pathological changes, analyzing the relation between the MR texture parameters and the TPE fluorescence intensities and the pathological changes and survivin expression of the tumor, and revealing implicit gene expression changes so as to provide an image index with higher evaluation prognosis sensitivity and specificity.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a magnetic nano-drug carrier and a preparation method thereof, and cytotoxicity experiments prove that the nano-drug carrier has good biocompatibility, good stability, targeting property and drug release property, and can be applied to preparation of cancer gene therapy drugs.

Drawings

FIG. 1 is a block diagram of a CA-SPIO-TPE-SP94-siRNA nano-drug carrier;

FIG. 2 is a high resolution Transmission Electron Microscope (TEM) of SPIO, CA-SPIO and CA-SPIO-TPE-SP94-siRNA (a-c), wherein (a) is an SPIO nanoparticle, (b) is a CA-SPIO nanoparticle, and (c) is a CA-SPIO-TPE-SP94-siRNA nanoparticle; (d) The particle size distribution bar graph of the SPIO nano particles is shown in the specification, (e) the particle size distribution bar graph of the CA-SPIO nano particles is shown in the specification, and (f) the particle size distribution bar graph of the CA-SPIO-TPE-SP94-siRNA nano particles is shown in the specification;

fig. 3 (a) is an infrared spectrum of SPIO nanoparticles, fig. 3 (b) is a photoluminescence spectrum of CA-SPIO-TPE nanoparticles, fig. 3 (c) is a thermogravimetric curve of CA-SPIO-TPE nanoparticles, fig. 3 (d) is a magnetic susceptibility curve of SPIO and CA-SPIO-TPE nanoparticles, fig. 3 (e) is an X-ray diffraction pattern of SPIO, and fig. 3 (f) is a Survivin siRNA release schematic of CA-SPIO-TPE-SP 94-siRNA;

FIG. 4 (a) is an in vitro T2 weighted MR image of aqueous solutions of different concentrations of CA-SPIO-TPE; FIG. 4 (b) is the transverse relaxation rate (R2) of aqueous solutions of different concentrations of CA-SPIO-TPE;

FIG. 5 (a) is a graph showing the CCK-8 results of CA-SPIO and CA-SPIO-TPE on Huh-7 cells; FIG. 5 (b) is a graph showing CCK-8 results of aqueous solutions of CA-SPIO-TPE-siRNA and CA-SPIO-TPE-SP94-siRNA at various concentrations against Huh-7 cells;

FIG. 6 (a) is a graph showing the results of staining dead/living cells with CA-SPIO-TPE, CA-SPIO-TPE-siRNA and CA-SPIO-TPE-SP94-siRNA particles; FIG. 6 (b) is a graph showing WB staining results of CA-SPIO-TPE, CA-SPIO-TPE-siRNA and CA-SPIO-TPE-SP94-siRNA particles on Huh-7 cells;

FIGS. 7 (a) and 7 (b) are fluorescence diagrams of CA-SPIO-TPE and CA-SPIO-TPE-SP94 particles in Huh-7 cells after being added to the cells for 4h and 12h, and the cells are detected by a confocal microscope; FIGS. 7 (c) and 7 (d) are fluorescent images of CA-SPIO-TPE and CA-SPIO-TPE-SP94 particles in Huh-7 cells after being added to the cells for 4h and 12h, and the cells are detected by a flow cytometer; FIG. 7 (e) is an average fluorescence plot of CA-SPIO-TPE, CA-SPIO-TPE-SP94 particles cultured for 4h and 12h in Huh-7 cells;

FIG. 8 (a) is a fluorescence image of nude mice 24 hours after implantation of tumors by intravenous injection of free CA-SPIO-TPE and CA-SPIO-TPE-SP94 nude mice; FIG. 8 (b) is a fluorescence imaging image of nude mice major organs CA-SPIO-TPE and CA-SPIO-TPE-SP94 transplanted tumors 24 hours after intravenous injection of free CA-SPIO-TPE and CA-SPIO-TPE-SP94 transplanted tumors; FIG. 8 (c) is a graph showing the total fluorescence intensity of tumor sites of nude mice quantitatively within 24 hours; FIG. 8 (d) is a graph of magnetic resonance imaging of CA-SPIO-TPE-SP94 and CA-SPIO-TPE at various time points before and after intravenous injection of Huh-7 tumor-bearing mice; FIG. 8 (e) is a graph of quantitative analysis of tumor T2 reduction; p < 0.05: < p < 0.01);

FIG. 9 (a) is a graph showing tumor growth in each group of tumor-bearing mice; FIG. 9 (b) is a graph showing the body weight of each group of tumor-bearing mice; FIG. 9 (c) is a graph showing the survival rate of each group of tumor-bearing mice; FIG. 9 (d) is a tumor development of day 14; FIG. 9 (e) is a HE staining pattern of tumors and internal organs of each group of tumor-bearing mice.

Detailed Description

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.

EXAMPLE 1 preparation of quaternary ammonium Cationized Amylose (CA)

Weighing 0.50g of amylose, adding the amylose into 25mL of distilled water, adjusting pH to be 12-14 by using 4mol/L NaOH solution, and heating and stirring to disperse the amylose; slowly dropwise adding 5mL of aqueous solution containing 0.25g of active etherifying agent GTA, and continuously stirring at 50 ℃ for reaction for 12 hours; after the reaction, the pH value is regulated to be neutral by using a hydrochloric acid solution, all the solution is transferred into a cellulose dialysis bag with the molecular weight cut-off of 8000-14000 Da, the solution is dialyzed for 3 days by using deionized water, and the dialysis solution is filtered and freeze-dried to obtain quaternary ammonium cationic amylose, which is marked as CA.

Example 2 aqueous solution of a combination of a quaternary ammonium cationized amylose and a superparamagnetic iron oxide nanoparticle Preparation of (CA-SPIO)

Weigh 0.20g of quaternary ammonium cationized amylose in a 50mL flask, add 20mL of distilled water and stir to dissolve it; weigh 0.20g FeCl ₃ ·6H ₂ O and 0.10g FeCl ₂ ·4H ₂ O was dissolved in 5mL of distilled water, and added to the above system, followed by stirring with nitrogen for 30 minutes. The flask was placed in a water bath at 80℃and 2.5mL of 25% aqueous ammonia was added with vigorous stirring using a syringe and reacted for 1 hour. After the reaction is finished, the temperature is reduced to room temperature, and the whole solution is transferred into a cellulose dialysis bag with the molecular weight cutoff of 8000-14000 Da for dialysis for 2 days. After insoluble substances are removed by centrifugation of the dialysate, the supernatant is preserved at 4 ℃ to obtain an aqueous solution of a complex of quaternary ammonium cationic amylose and superparamagnetic nanoparticles (Superparamagnetic iron oxide, SPIO), designated CA-SPIO.

EXAMPLE 3 quaternary ammonium cationized amylose-tetraphenylethylene-superparamagnetic iron oxide nanoparticle (CA- SPIO-TPE) is prepared

Weigh 4mg TPE in 4mL CH ₂ Cl ₂ Adding 20mg of aqueous solution (CA-SPIO) of the quaternary ammonium cationic amylose and superparamagnetic nanoparticle complex, uniformly mixing at room temperature, dripping the mixture into 40mL of pure water while ultrasonic treatment for 1 hour, transferring the product into a dialysis bag with the molecular weight cutoff of 2000Da, dialyzing for 3 days, changing the pure water for 2 times per day, freeze-drying the dialyzate, and collecting the solid product for later use at 4 ℃ to obtain quaternary ammonium cationic amylose-tetraphenyl ethylene-superparamagnetic iron oxide nanoparticles which are marked as CA-SPIO-TPE.

EXAMPLE 4 targeting of SP94 targeting peptide modified quaternary ammonium cationic amylose-tetraphenylethylene-superparamagnetic oxygen Iron melting nano particlePreparation of aqueous solutions of the granules (CA-SPIO-TPE-SP 94)

Weigh 20mg of the short peptide of S94 in 4mL DMSO and stir for about 1 hour to dissolve; 40mg of EDC. HCl and 20mg of NHS were then added and stirred at room temperature in the dark for 4 hours to give a DMSO solution of the SP94 targeting peptide active ester. An aqueous solution of CA-SPIO-TPE (containing 0.10g of CA) prepared in example 3 was taken in a 25mL round bottom flask, glacial acetic acid was added dropwise to adjust the pH to about 5, and then the DMSO solution of the SP94 targeting peptide active ester was added and reacted at room temperature in the absence of light for 48 hours. After the reaction, the reaction solution is completely moved into a cellulose dialysis bag with the molecular weight cut-off of 8000-14000 Da for dialysis for 3 days. And centrifuging the dialyzate to remove insoluble substances, and preserving the upper layer liquid at 4 ℃ in a dark place to obtain an aqueous solution of SP94 targeting peptide targeting modified quaternary ammonium cationic amylose-tetraphenylethylene-superparamagnetic iron oxide nano-particles, which is marked as CA-SPIO-TPE-SP94.

Example 5 preparation of CA-SPIO-TPE-siRNA and CA-SPIO-TPE-SP94-siRNA nano drug Carrier

The molar ratio of N/P (wherein N is derived from CA-SPIO-TPE and P is derived from siRNA) is 5:1 was diluted with distilled water and then an equal amount of siRNA solution (2 pmol/. Mu.L) was added. The mixture was spun for 5 seconds and then left at room temperature for 20 minutes to form the CA-SPIO-TPE-siRNA nano drug carrier.

The molar ratio of N/P (wherein N is derived from CA-SPIO-TPE-SP94 and P is derived from siRNA) is 5:1 was diluted with distilled water and then an equal amount of siRNA solution (2 pmol/. Mu.L) was added. The mixture was spun for 5 seconds and then left at room temperature for 20 minutes to form the CA-SPIO-TPE-SP94-siRNA nano-drug carrier. The structure is shown in figure 1.

Example 6 cell experiments and animal experiments

1. Cell culture and animal culture

1.1 cell culture

Complete medium was prepared and 10% FBS, 1% diabody was mixed in DMEM medium. Huh-7 human hepatocellular carcinoma cells were placed in a petri dish containing complete medium and incubated at 37℃with 5% CO ₂ Culture in saturated humidity incubatorCulturing, and taking cells in logarithmic growth phase for experiment after 2-3 days of passage.

1.2 animal culture

BALB/c nude mice are adopted, male, age and age are about 4 weeks (28-34 days), average weight is about 15-18 g, the nude mice are placed in SPF environment with temperature of about 22-26 ℃ and humidity of about 40-60%, free feeding and drinking water are carried out, SPF grade mouse feed, distilled water, cages and the like are taken, padding is replaced regularly, drying and cleaning are kept, and the nude mice are adaptively fed for 7 days and then used for experiments. The experiment complies with the welfare and ethical guidelines of experimental animals, is approved by the committee for experimental animal management and use of the university of Zhongshan, and is approved by the ethical examination consent approval number SYSU-IACUC-2020-000048 of the university of Zhongshan.

1.3 tumor modeling

Taking Huh-7 human hepatocellular carcinoma cells in logarithmic growth phase, adding trypsin liquid into adherent cells, digesting and resuspension to form cell suspension, centrifuging the cell suspension at 1000 rpm for 5 min, discarding upper liquid, resuspension lower cell in PBS, and regulating cell concentration to about 1×10 ⁷ And each mL. Taking a nude mouse, injecting and anaesthetizing the nude mouse by using 10% chloral hydrate to the abdominal cavity, slowly injecting and injecting a cell suspension into the inguinal space on the right side in a subcutaneous mode, inoculating liquid with the volume of about 0.2mL, periodically observing the nude mouse, and observing that the inguinal space of the nude mouse has small tumor formation (the maximum meridian of the tumor is less than 10 mm) after about 5 days, so that the nude mouse is successfully molded, and can be used for further experiments.

2. Structural characterization test

2.1 Infrared (FTIR) test

Adopts a potassium bromide tabletting method, and adopts a Fourier infrared (Fourier transform infrared, FTIR) spectrometer to respectively measure that the SPIO nano micelle of the superparamagnetic nano particles is 4000-399 cm ^-1 Infrared spectrum in the range.

2.2X-ray diffraction Spectrometry (XRD) testing

The freeze-dried sample is spread on a sample plate, and the crystal structure of the freeze-dried sample is tested by an X-ray diffractometer. Under the condition of 40kV/30mA, the scanning speed is 10 degrees/min, the scanning angle is 20-70 degrees, and the Cu target is K alpha rays )。

2.3 high resolution Transmission Electron Microscopy (TEM) testing

The CA-SPIO and CA-SPIO-TPE nano-micelles dissolved in normal hexane are sufficiently diluted to 0.5mg/mL, then a sample is dripped on a copper mesh with an amorphous carbon film coated on the surface, and the copper mesh is dried in air and then placed in a high-resolution transmission electron microscope (Transmission electron microscopy, TEM) to observe the overall morphology and particle size distribution of the nano-particles.

2.4 particle size distribution and Zeta potential test

The particle size distribution of the polymer micelles was measured by dynamic light scattering (Dynamic light scattering, DLS), the particle size, particle size distribution and Zeta potential of the nanoparticles were measured using a particle size and Zeta potential analyzer, the test temperature was 25 ℃, each sample was measured 3 times, and the average was taken.

2.5 fluorescence Spectrum testing

The nano particles are dissolved in acetonitrile, nano micelle solution of 1mg/mL is prepared, a series of acetonitrile/water composite micelle solutions are prepared according to gradient, and the scanning wavelength is 350 nm-600 nm by utilizing a fluorescence spectrophotometer for testing.

2.6 magnetic Property testing

And taking a proper amount of freeze-dried sample, and testing the hysteresis curve of the magnetic nano composite particles by using a magnetic property measurement system. The temperature is 300K, and the magnetic field intensity is within the range of +/-20000 Oe.

2.7 thermal weight loss (TGA) test

And testing a proper amount of freeze-dried sample by using a thermogravimetric analyzer (Thermogravimetry analysis), wherein the heating rate is 20 ℃/min, the purge gas is 40mL/min of nitrogen, the shielding gas is 20mL/min of nitrogen, and the scanning temperature range is 40-700 ℃.

2.8 Magnetic Resonance Imaging (MRI) testing

An aqueous solution of CA-SPIO-TPE material was prepared at 0.5mg/mL (Fe concentration 1.43 mM), then serially diluted to 8 different concentration gradients, with concentrations ranging from 1.43, 0.72, 0.36, 0.19, 0.09, 0.04, 0.02, 0.01mM [ Fe ], each filled with 1.5mL of solution using 8 2mL clear rigid plastic centrifuge tubes, sequentially placed on foam plates, and scanned with a 1.5T MRI scanner at room temperature. The MRI scan sequence includes T2WI, T2 mapping.

The T2WI parameters are as follows: repetition Time (TR)/Echo Time (TE) =2600 ms/100ms; flip angle (Flip angle) =90°; NA (Number of acquisitions) =6; acquisition matrix (Acquisition matrix) =384×305; FOV (Field of view) =80 mm×80mm; layer thickness (Slice thickness) =2mm; interlayer spacing (Slice gap) =2mm. T2 mapping adopts single-layer multi-echo self-selection echo sequence scanning, and the parameters are as follows: tr=1500 ms, te=0, 20, 40 … 160ms, na= 3,Acquisition matrix =176×123mm, fov=80 mm× 80mm,Slice thickness =1.5 mm, slice gap=1.5 mm.

3. In vitro experiments

3.1 cytotoxicity test

The experiment selects Huh-7 cells as a material toxicity evaluation cell model, and adopts a method of detecting cell activity by CCK-8 to evaluate the toxicity of 4 nano particles of materials CA-SPIO, CA-SPIO-TPE-siRNA and CA-SPIO-TPE-SP94-siRNA to the Huh-7 cells.

The specific operation steps are as follows: huh-7 cells were first seeded into 96-well plates at a density of 5000 cells/well and then placed in a carbon dioxide incubator for culture adherence overnight. Subsequently, the original medium was aspirated, and replaced with fresh complete medium containing different concentrations of material, the concentration of material selected being set to 1000. Mu.g/mL, 500. Mu.g/mL, 250. Mu.g/mL, 100. Mu.g/mL, 10. Mu.g/mL (polymer concentration); there were 5 parallel groups of 2000 μg/mL, 1000 μg/mL, 500 μg/mL, 250 μg/mL, 125 μg/mL (micelle concentration) per concentration. The cells were then incubated in the incubator for 24 hours, washed once with PBS after incubation and 100. Mu.L of fresh medium (containing 10% CCK-8) was added to each well. Incubation in incubator for a period of time, and finally detection and recording of absorbance at 450nm wavelength using a microplate reader, cell viability was calculated by the following formula: cell viability (%) = (experimental group absorbance-blank group absorbance)/(negative control group absorbance-blank group absorbance) ×100%.

3.2 laser confocal test

The distribution of nanoparticles in cells was examined using a laser confocal microscope. Intracellular fluorescence detection was performed using CA-SPIO-TPE and CA-SPIO-TPE-SP94 nanoparticles. The specific method comprises the following steps: soaking the cover glass in alcohol, performing ultrasonic treatment, cleaning, drying, sterilizing at 121 ℃, and drying again for later use. Aseptically placing a sterile cover slip into a 35mm culture dish to prepare a cell suspension 10 ⁵ Each well was filled with 2 mL/mL, and cells were allowed to slide on the coverslip. After 24 hours of incubation, the original whole medium was replaced with a complex micelle solution containing CA-SPIO-TPE and CA-SPIO-TPE-SP94 nanoparticles. After culturing for a prescribed period of time (4 hours and 12 hours), all the medium was removed, a 4% paraformaldehyde in PBS was added, and the cells were fixed in an incubator for 30 minutes. After 30 minutes, the paraformaldehyde solution was removed, washed 1 time with PBS solution, DAP in PBS solution (DAPI concentration 2 g/mL) was added, and stained in an incubator for 10 minutes. After 10 minutes, the DAPI PBS solution was removed, all solutions were removed, and the solution was washed twice with PBS solution. Then picking up the cover glass which is full of cells, back-buckling on a clean glass slide, sealing the glass slide with glycerol, fixing the nail polish, and finally preserving in a dark place at 4 ℃. The prepared cell slide was observed under a confocal laser microscope, where the dimming parameter of DAPI was (excitation light wavelength/emission light wavelength: 405nm/453 nm), and where the dimming parameter of TPE was (excitation light wavelength/emission light wavelength: 488nm/523 nm).

3.3 flow cytometer testing

And (3) utilizing a flow cytometer to quantitatively target the promotion effect of the nano micelle on cell phagocytosis. The specific operation is as follows: preparing different cells into suspension with concentration of 10 ⁵ Each mL was inoculated into 2mL of cell suspension per dish in 35mm bottom diameter dishes. After 24 hours of incubation, the original whole medium was replaced with a complex micelle solution containing CA-SPIO-TPE and CA-SPIO-TPE-SP94 nanoparticles. The culture was further carried out in an incubator for 4 hours and 12 hours, respectively.

4. In vivo experiments

4.1 in vivo fluorescence imaging test

The drug was injected by tail vein injection and CA-SPIO-TPE-SP94 nanoparticles were fluorescence imaged using a small animal living imaging system to explore the tumor/organ distribution and body clearance rate of TPE fluorescent nanoparticles in vivo. Nude mice bearing Huh-7 tumor were injected with CA-SPIO-TPE and CA-SPIO-TPE-SP94 nanoparticles, respectively, and fluorescence images were collected 1 hour, 6 hours, 12 hours, and 24 hours after injection.

4.2 in vivo magnetic resonance imaging testing

The drug was injected by tail vein injection and the effect of CA-SPIO-TPE-SP94-siRNA and CA-SPIO-TPE-siRNA on tumor signal changes was investigated using small animal MRI scans. After anesthesia with isoflurane gas, tumor bearing nude mice were scanned at room temperature using a 3.0T MRI scanner with small animal specific coils, the scanning sequence included T2 SPIR (Spectral presaturation with inversion recovery), T2 mapping.

The T2WI SPIR parameters are as follows: TR/te=1600 ms/60ms; flip angle = 90 °; na=3;

acquisition matrix =252×254; FOV = 128mm x 128mm; slice thickness=1 mm; slice gap=1 mm. T2 mapping adopts single-layer multi-echo self-selection echo sequence scanning, and the parameters are as follows: tr=1400 ms, te=25, 50, 75, 100ms, na= 3,Acquisition matrix =109×108mm, fov=128 mm× 128mm,Slice thickness =1.5 mm, slice gap=1.5 mm.

4.3 in vivo tumor suppression experiments and histopathological examination

Tumor-bearing nude mice were randomly divided into different experimental groups of 3 mice each, and were dosed at 5mg/kg on days 0, 2, 4, with 200 μl of each mouse tail intravenously, for a total of three doses. The tumor volume of the nude mice was measured daily with a vernier caliper, and the body weight of the nude mice was weighed with an electronic scale. Tumor-bearing nude mice were sacrificed after the end of the experiment and complete tumors were removed for photography. Tumor volume = 0.5 x length x width. Tumor-bearing nude mice survival curve experiments were performed with 4 mice per group. The tumor-bearing nude mice are randomly divided into different experimental groups, 3 nude mice in each group are dosed according to the dose of 5mg/kg on days 0, 2 and 4, 200 mu L of medicine is intravenously injected into each nude mouse tail, the tumor-bearing nude mice are sacrificed after the experiment on day 14 is finished, the heart, liver, spleen, lung, kidney and planted tumors of the animals are taken out, the animals are put into a 10% formaldehyde solution for dehydration, paraffin embedding and slicing are carried out, HE staining and sealing are carried out finally, and the structural change and damage condition of each viscera and tumor tissue are observed under a light microscope.

Results:

1. structural characterization test results:

after synthesis of the oleylamine and oleic acid stable hydrophobic SPIO, TPE is introduced into the SPIO nanoparticle core by way of physical entrapment through hydrophobic interactions due to both TPE and SPIO having hydrophobic character. Thus, CA-SPIO can introduce large amounts of hydrophobic TPE and SPIO into the core while maintaining water solubility with the hydrophilic outer shell of CA. High resolution Transmission Electron Microscopy (TEM) pictures show that SPIO nanoparticles are black dots with average sizes of 5-8nm, and CA-SPIO-TPE-SP94-siRNA nanoparticles are spherical, and the average sizes are 155nm and 178nm respectively (refer to FIG. 2). The PDI of the CA-SPIO and CA-SPIO-TPE-SP94-siRNA nanoparticles was 0.18 and 0.25, respectively. The size of the nanoparticles is between 100nm and 200nm, the best choice for enhancing the permeability and retention. The surface potentials of CA-SPIO and CA-SPIO-TPE-SP94-siRNA were 23.5mV and 5.8mV, respectively, indicating successful adsorption of negatively charged siRNA.

The infrared spectrum of the SPIO nanoparticle is shown in FIG. 3 (a), 560.0cm ^-1 The peak at this point is the stretching vibration absorption peak of the metal atom and the oxygen atom. 1570cm ^-1 The two peaks at which correspond to the symmetrical stretching vibration peak and the antisymmetric stretching vibration peak of c=o, which are characteristic peaks of carboxylate salts. 2920cm ^-1 And 2850cm ^-1 The two peaks at this position correspond to the C-H stretching vibration absorption peaks in the oleic acid molecule.

Photoluminescence (PL) spectra of CA-SPIO-TPE (25-1000 μg/mL) are shown in fig. 3 (b), with an absorption peak at 430nm indicating that TPE has been successfully incorporated into CA-SPIO.

FIG. 3 (c) shows the thermogravimetric curve of CA-SPIO between 40℃and 700 ℃. The final mass loss of CA-SPIO-TPE was 63%, i.e. the SPIO content was 37%. Due to the addition of the SPIO, the content of the thermally degradable component in the CA-SPIO is reduced, which indicates that the SPIO is successfully introduced into the CA-SPIO. For magnetic materials, magnetic response is one of the important performance indicators for detecting magnetic materials. Hysteresis loops are commonly used to characterize the response of a magnetic material to an external magnetic field.

Fig. 3 (d) shows hysteresis loops of SPIO and CA-SPIO at 300K. In the cyclic scan of the applied magnetic field from-20000 oE to 20000oE, the remanent magnetization of SPIO or CA-SPIO-TPE is zero when the applied magnetic field strength is zero. This result indicates that both SPIO and CA-SPIO-TPE have superparamagnetism. The saturation magnetization of SPIO and CA-SPIO-TPE was 53.8emu/g and 26.3emu/g, respectively. The reason for the reduced saturation magnetization of CA-SPIO is that CA wraps around the surface of SPIO, resulting in reduced magnetization. The CA-SPIO-TPE has good magnetic response to an external magnetic field and can be used for subsequent MRI scanning.

As shown in fig. 3 (e), XRD results confirmed SPIO to be a superparamagnetic nanoparticle. The characteristic peaks are at 30.2o, 35.6o, 43.0o, 53.4o, 57.1o and 62.5o, corresponding to (220), (311), (400), (422), (511) and (440) of spinel, respectively.

As shown in FIG. 3 (f), the CA-SPIO-TPE-SP94-siRNA nano-drug carrier has good drug release effect of Survivin siRNA at 37 ℃, 50.0% of Survivin siRNA is released after 5 hours, and 79.8% is released after 24 hours.

2. In vitro T2 weighted imaging

As shown in fig. 4, T2-weighted imaging uses a conventional spin echo SE sequence scan with pulse repetition interval tr=1500 ms, echo times te=96.191, 167.98,293.32,487.83,752.57,1032.6,1259.3,1430.2ms, fs=1.5. As the Fe concentration increases, the MR image becomes darker. The Fe (Mm) concentration corresponds to the inverse of the T2 relaxation time, 1/T2 (s-1). The slope of the line fitted by the data points is the T2 relaxation efficiency, r2=39.1 mm ^-1 s ^-1 . This indicates that the aqueous solution of CA-SPIO-TPE has excellent magnetic resonance imaging performance.

3. In vitro cytotoxicity test results

1) And detecting cytotoxicity of the CA-SPIO, CA-SPIO-TPE-SP94-siRNA and the CA-SPIO-TPE-siRNA nano particles by adopting a CCK8 colorimetric method.

As shown in FIG. 5 (a), the concentration of the aqueous solution in CA-SPIO was from 10 to 1000. Mu.g mL ^-1 In the range of (2), the cell viability was about 85%, indicating that CA-SPIO and CA-SPIO-TPE nanoparticles have good biology in HUH-7 cellsCompatibility.

In addition, as shown in fig. 5 (b), CA-SPIO-TPE-SP94-siRNA and CA-SPIO-TPE-siRNA nanoparticles showed good inhibition effect on Huh-7 cells at low concentration. The half maximal inhibitory concentrations (IC 50 values) of CA-SPIO-TPE-siRNA and CA-SPIO-TPE-SP94-siRNA nanoparticles after 24h incubation with Huh-7 cells were 78.6 μg/mL and 29.4 μg/mL, respectively. The inhibition effect of the CA-SPIO-TPE-SP94-siRNA nanoparticle is superior to that of the other two groups, which indicates that targeting the SP94 peptide can enhance accumulation of Survivin siRNA in cells.

2) Live/dead staining analysis:

as shown in FIG. 6 (a), the inventors found that CA-SPIO, CA-SPIO-TPE nanoparticles maintained good cell viability for Huh-7 cells after 48h of culture, since almost all samples showed green fluorescent signals (living cells). Meanwhile, the CA-SPIO-TPE-siRNA and the CA-SPIO-TPE-SP94-siRNA nano particles have good inhibition effect on Huh-7 cells, and show a large amount of red fluorescent signals (dead cells). These results are consistent with those of CCK-8.

3) To investigate the mechanism of inhibition of Survivin siRNA on Huh-7 cells, protein expression levels of caspase-3 and Survivin in Huh-7 cells were examined by Western blotting.

As shown in FIG. 6 (b), the expression level of caspase-3 was significantly up-regulated after 48h of treatment of Huh-7 cells with CA-SPIO-TPE-siRNA nanoparticles, indicating that Survivin siRNA successfully inhibited tumor cell growth. Caspase-3 plays an irreplaceable role in apoptosis. The overexpression of Caspase-3 shows that CA-SPIO-TPE nano particles carrying Survivin siRNA can remarkably inhibit the growth of Huh-7 cells. Meanwhile, the down regulation of Survivin protein shows that the CA-SPIO-TPE-siRNA nanoparticle has a successful RNA interference effect.

4. In vitro cell uptake studies: the intracellular distribution of the CSP intracellular fragment was examined by Confocal Laser Scanning Microscopy (CLSM).

As shown in fig. 7 (a), after 4 hours of incubation with micelles, the intensity of red in the cytosol of SP94 peptide-targeted micelles incubated with non-targeted micelles was significantly higher than that of non-targeted micelles, indicating that the targeted micelles could preferentially accumulate in the cytoplasm by receptor-mediated endocytosis processes.

Meanwhile, as shown in fig. 7 (b), the difference in fluorescence intensity between the SP94 peptide-targeted micelle and the non-targeted micelle gradually expands after 12 hours of incubation, because the targeting effect of the SP94 peptide increases with the passage of time, and more cellular uptake occurs after 12 hours.

The fluorescence results of the flow cytometry at 4h (fig. 7 (c)) and 12h (fig. 7 (d)) also demonstrate active targeting of the SP94 peptide. In addition to SP94 promoting receptor-mediated uptake, the cationic surface of CA-SPIO-TPE-siRNA nanoparticles is tightly associated with anionic cell membranes by nonspecific electrostatic interactions. Nanoparticles can enter mammalian cells by endocytosis (Conner & Schmid 2003) and then form membrane-bound vesicles and encapsulate the nanoparticles for internalization.

5. Living AIE imaging and biodistribution

To investigate whether CA-SPIO-TPE nanoparticles were suitable for mouse tumor imaging, 1mg mL-1 of CA-SPIO-TPE nanoparticles and CA-SPIO-TPE-SP94 nanoparticles were intravenously injected into BALB/c mice bearing Huh-7 tumors, respectively. Both CA-SPIO-TPE and CA-SPIO-TPE-SP94 accumulated in the tumor within 1h after injection. The fluorescence signal in the tumors was gradually increased to the highest brightness at 6h (FIG. 8 (a)), suggesting that CA-SPIO-TPE and CA-SPIO-TPE-SP94 have good in vivo imaging ability for Huh-7 tumors. Mice were sacrificed 24h after injection and the distribution of CA-SPIO-TPE and CA-SPIO-TPE-SP94 in tumors and different organs was observed.

As shown in fig. 8 (b), the prepared nanoparticles flowed in the circulatory system of the mice and accumulated mainly in the liver and tumors. This may be due to the metabolic function of the animal. Tumor mean fluorescence intensity results showed that the AIE fluorescence intensities within 6h after injection were similar for the CA-SPIO-TPE group and the CA-SPIO-TPE-SP94 group. After 6 hours, the fluorescence intensity of the CA-SPIO-TPE-SP94 group is higher than that of the CA-SPIO-TPE group. The average fluorescence intensities of the two groups were significantly different. This indirectly reflects the aggregation of the two sets of TPEs.

6. In vivo magnetic resonance imaging

Since SPIO is able to produce a low signal on T2WI, tumor targeting of CA-SPIO-TPE-SP94 can be monitored non-invasively by MRI.

The invention evaluates the imaging efficiency of MRI in vivo by using a Huh-7 cell-derived subcutaneous transplantation tumor mouse model. Mice were scanned prior to injection of CA-SPIO-TPE or CA-SPIO-TPE-SP 94. After each mouse was injected with the prepared nano-drug carrier, scanning was performed before injection, 6h after injection, 12h after injection, and 24h after injection, respectively. Representative scanned images of the same tumor slice are displayed at different time points (fig. 8 (c)). After injection of CA-SPIO-TPE-SP94, the normalized MR signal intensity values and T2 values of the tumor region of interest were significantly reduced at 6h and 12h after injection. After injection of CA-SPIO-TPE, the normalized MR signal intensity value and T2 value of Huh-7 tumor have no obvious change. The tumor interest T2 values of the CA-SPIO-TPE group and the CA-SPIO-TPE-SP94 group were reduced to 5%/9% and 2%/34% (p < 0.003) respectively at 6h/12h after injection. 24 hours after injection, the T2 values of the CA-SPIO-TPE group and the CA-SPIO-TPE-SP94 group were reduced to 16% and 4% (P > 0.05), respectively (FIG. 8 (d)). These results are consistent with the discovery that SP94 modified nanocarriers have higher targeting ability for Huh-7 cells (fig. 7). Using an active targeted delivery system, MR contrast signals can be significantly improved. MRI scanning provides more valuable detail than fluorescence imaging. First, MRI shows better spatial resolution than fluorescence imaging and may not be limited by penetration depth. Second, cross-sectional MR images provide more information about nanocarrier distribution and concentration in tumors than just two-dimensional information about fluorescence imaging metabolism. Finally, the heterogeneous distribution of low signal intensities on the T2 weighted image reflects tumor heterogeneity, with intra-tumor lamellar T2 low signals likely corresponding to more active tumor cells or better perfused areas (fig. 8 (e)).

7. Anti-tumor effect in vivo

The anti-tumor effect of CA-SPIO in nude mice bearing human Huh-7 is studied.

As shown in FIG. 9 (a), tumors treated with PBS, CA-SPIO and CA-SPIO-TPE showed rapid growth of tumor at day 14, with average tumor sizes of 825, 574 and 462mm, respectively ³ . At the same time, tumors treated with Survivin siRNA and CA-SPIO-TPE-SP94-siRNA were slightly inhibited on day 14 with average tumor sizes of 272 and 253mm, respectively ³ . The CA-SPIO-TPE-SP94-siRNA can obviously inhibit the growth of tumor, and the average size of the tumor on day 14 is 27.5mm ³ . The single injection of the CA-SPIO-TPE-SP94-siRNA can completely inhibit the growth of tumors, and no recurrence exists, which indicates that the small-dose siRNA and SP94 treatment can effectively release the siRNA and perform targeted gene therapy, thereby achieving the purpose of radically curing tumors. This finding is consistent with in vivo fluorescence and magnetic resonance imaging results. In addition, weight loss was also analyzed to indicate toxicity caused by treatment (fig. 9 (b)). The weights of all the nano-film treated groups of nude mice slightly increased, but were not significantly different from the control group (PBS group) (P > 0.05), and the weights of all the groups of nude mice slightly increased (P < 0.05), but were not significantly different from the control group (PBS group). The siRNA dose used in the treatment was well tolerated. Meanwhile, the survival rate of the nude mice injected with the CA-SPIO-TPE-SP94-siRNA is highest. H of major organs &The E-stained image further showed that all treatments containing siRNA were biocompatible and safe for nude mice (fig. 9 (E)). The CA-SPIO combined siRNA treatment combines real-time tracing, controllable siRNA release and gene therapy, and realizes the optimal strategy of anti-tumor treatment with low side effect.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A magnetic nano-drug carrier, which is characterized by comprising a coupling product of SP94 targeting peptide and siRNA delivery carrier, wherein the coupling product of the siRNA delivery carrier comprises superparamagnetic iron oxide nanoparticles, quaternary ammonium cationized amylose and tetraphenylethylene coupled to form quaternary ammonium cationized amylose-tetraphenylethylene-superparamagnetic iron oxide nanoparticles, and the siRNA is adsorbed in the magnetic nano-drug carrier; the particle size of the superparamagnetic iron oxide nanoparticle is 5-8 nm; the particle size of the magnetic nano-drug carrier is 100-200 nm; the surface potential of the magnetic nano-drug carrier is 5.8mV, and the polymer dispersibility index of the magnetic nano-drug carrier is 0.25;

The preparation method of the magnetic nano-drug carrier comprises the following steps:

s2, dissolving tetraphenyl ethylene in CH ₂ Cl ₂ Adding the quaternary ammonium cationic amylose-superparamagnetic iron oxide nanoparticle aqueous solution obtained in the step S1, uniformly mixing to form a mixture II, dripping the mixture II into pure water in ultrasound, dialyzing, freeze-drying, and collecting a solid product to obtain the quaternary ammonium cationic amylose-tetraphenylethylene-superparamagnetic iron oxide nanoparticle;

2. The magnetic nano-drug carrier according to claim 1, wherein the preparation method of the quaternary ammonium cationized amylose comprises the following steps:

3. The magnetic nano-drug carrier according to claim 1, wherein the concentration of ammonia water in the step S1 is 25% by mass.

4. The magnetic nano-drug carrier according to claim 1, wherein the molecular weight of the dialysis trapping mixture I in the step S1 is 8000-14000 Da; the molecular weight of the dialysis interception mixture II in the step S2 is 2000Da; the molecular weight of the dialysis interception mixture III in the step S3 is 8000-14000 Da.

5. The magnetic nano-drug carrier of claim 1, wherein the molar ratio of nitrogen in the aqueous solution of SP94 targeting peptide-targeted modified quaternary ammonium cationized amylose-tetraphenylethylene-superparamagnetic iron oxide nanoparticle to phosphorus in the aqueous solution of siRNA in step S4 is 5:1.

6. Use of the magnetic nano-drug carrier according to any one of claims 1-2 in the preparation of cancer gene therapy drugs.