CN106039324B

CN106039324B - A kind of biomimetic type magnetic corpusculum and preparation method thereof loading siRNA

Info

Publication number: CN106039324B
Application number: CN201610547657.9A
Authority: CN
Inventors: 谢海燕; 张帆; 马光辉; 魏炜; 赵龙剑
Original assignee: Institute of Process Engineering of CAS; Beijing Institute of Technology BIT
Current assignee: Institute of Process Engineering of CAS; Beijing Institute of Technology BIT
Priority date: 2016-07-12
Filing date: 2016-07-12
Publication date: 2019-08-30
Anticipated expiration: 2036-07-12
Also published as: CN106039324A

Abstract

The present invention relates to a kind of biomimetic type magnetic corpusculums and preparation method thereof for loading siRNA, belong to chemistry and field of biomedicine.For the biomimetic type magnetic corpusculum using water-soluble MNC as core, outsourcing leucocyte film is mounted with siRNA between MNC and leucocyte film；Outside the biomimetic type magnetic corpusculum leucocyte film can covalent coupling have antibody or peptide with modification group.By preparing MNC, M, MNC:siRNA, MNC:siRNA solution, M solution and buffer are mixed, the biomimetic type magnetic corpusculum is obtained；By modification group by peptide or antibody modification to the leucocyte film of the biomimetic type magnetic corpusculum, the biomimetic type magnetic corpusculum of coupled antibody or peptide is obtained.MNC in the biomimetic type magnetic corpusculum has positive charge, and adsorbable siRNA, outside cladding M is bionical, can efficiently, be rapidly achieved lesion, while avoiding the immune clearance of organism.

Description

A kind of biomimetic type magnetic corpusculum and preparation method thereof loading siRNA

Technical field

The present invention relates to a kind of biomimetic type magnetic corpusculums and preparation method thereof for loading siRNA, belong to chemistry and biomedicine Field.

Background technique

RNA interference (RNAi) refers in biological cell, and the exogenous or endogenous double-strand that targeting target gene is homologous Cause silencing specific genes after tiny RNA (siRNA) inducible transcription.RNAi class drug table in the illnesss such as tumour, genopathy Reveal great application prospect, but up to the present, RNAi technology still has siRNA targeting and taken off in terms for the treatment of tumour The problems such as targeted effect, internal unstable, interferon effect, drug delivery system, administration mode and drug safety.

The gene transfection that nano-carrier mediates has the unexistent advantage of other drugs carrier, especially magnetic nano-particle Possess good biocompatibility, superior magnetic performance and higher stability, can be used for magnetic targeted siRNA conveying. Superparamagnetism Fe₃O₄Nano material has outside plus under magnetic field condition compared with ferromagnetism, and magnetism disappears quickly after removing externally-applied magnetic field, makes Obtaining nanoparticle will not be permanently magnetized in magnetic field, and this unique physical and magnetism characteristic can be used as the target of siRNA drug To carrier, under magnetic fields, targeting conveying drug to the lesions positions such as tumour.

Under certain temperature, the threshold value that superparamagnetic transformation occur in ferromagnetic particles is normal by Effective Magnetic Anisotropy and Boltzmann Number determine and it is immutable, the stability of magnetic moment and the particle can be enhanced in the partial size for increasing ferromagnetic particles, but simultaneously The particle is resulted in from superparamagnetism to ferromagnetic transformation, thus caused remanent magnetism causes the particle that cannot be dispersed in molten In liquid, the efficiency and repeatability of bio-separation are seriously reduced.Therefore, how to ensure superparamagnetism and high ratio saturated magnetization While intensity, enhance magnetic moment and magnetic navigability, improve stability and guarantee good water solubility, is existed using magnetic separation technique Critical issue urgently to be resolved in field of biomedicine practical application.

In addition, siRNA anti-tumor drug needs to arrive at diseased region by blood circulation in biologic artifact, to play Therapeutic effect.The design object of super-paramagnetism nano pharmaceutical carrier first is that when maintaining its long circulating in biological organic body Between.Its long circulating inside biologic artifact can be realized by modify or be modified to carrier system surface, it is big at present Multi-pass crosses surface modification, realizes carrier in biology such as polyethylene glycol (polyethylene glycol, PEG) or other modifications Long circulating inside organism has cytotoxicity although extending circulation time more.

Summary of the invention

It is directed in the prior art, the unstability of siRNA anti-tumor drug, easily by RNA enzyme degradation, easily by body The defect that kidney organ removes and the circulating half-life of internal body is extremely short, one of the objects of the present invention is to provide a kind of dresses Carry siRNA biomimetic type magnetic corpusculum, the biomimetic type magnetic corpusculum can efficiently, be rapidly gathered in cell, reach tumor focus portion Position, while the bodies obstacles such as the immune clearance of biologic artifact immune system, the phagocytosis of macrophage are avoided, reach oncotherapy Good result.

The second object of the present invention is to provide a kind of preparation method of biomimetic type magnetic corpusculum for loading siRNA, through described The biomimetic type magnetic corpusculum for the loading siRNA that method can be prepared.

The third object of the present invention is to provide a kind of method of modifying of biomimetic type magnetic corpusculum for loading siRNA.

The purpose of the present invention is existing by following technical proposals.

A kind of biomimetic type magnetic corpusculum loading siRNA, the biomimetic type magnetic corpusculum is with water-soluble Fe₃O₄Nanocluster is core, Its outer cladding leucocyte film, in the Fe₃O₄SiRNA is mounted between nanocluster and leucocyte film；The Fe₃O₄Nanoclusters Cluster is by Fe₃O₄Nano crystal and polyethyleneimine (polyethyleneimine, PEI) composition, it is positively charged, partial size be 30nm~ 196nm。

Wherein, the preferably described Fe₃O₄The partial size of nanocluster is 81nm；

It is preferred that leucocyte film is the cell membrane of leukon J774A.1.

The biomimetic type magnetic corpusculum of the present invention for loading siRNA covalent coupling can also have with modification outside leucocyte film The antibody or peptide of group obtain the biomimetic type magnetic corpusculum of the loading siRNA of coupled antibody or peptide a kind of；

The antibody is the antibody that the end Fc has lysine residue；

The peptide is one or more of targeting peptides, fusogenic peptide and cell-penetrating peptide with amino；

The modification group is DBCO-PEG_n- NHS, n=1~2000, preferably n=4；

The modification group passes through the amino coupled in cycloalkyne modification and antibody or peptide.

A kind of preparation method for the biomimetic type magnetic corpusculum loading siRNA, steps are as follows for the preparation method:

Step 1: Fe₃O₄The preparation of nanocluster (referred to as MNC)

(1) under oxygen-free environment, NaOH is completely dissolved in diethylene glycol (Diethylene glycol, DEG), is made It is standby to obtain NaOH storing liquid；

Wherein, oxygen-free environment can be realized by using inert gas deoxidation treatment and protection；

Can be heated to 100 DEG C~150 DEG C is completely dissolved in NaOH in DEG, stops heating when NaOH is completely dissolved, preferably Being heated to 120 DEG C is completely dissolved in NaOH in DEG；

NaOH storing liquid can be maintained at 50 DEG C~100 DEG C and be stored for future use, NaOH storing liquid is preferably maintained at 70 DEG C Under store for future use；

It is preferred that NaOH storing liquid is prepared and is stored with the following method:

DEG is done into deoxidation treatment in the environment of inert gas, after oxygen is removed completely, NaOH is dissolved into DEG, It heats simultaneously, is warming up to 120 DEG C, stop heating when NaOH is completely dissolved, the preparation process whole process all needs inert gas Protection, obtaining pale yellow solution is NaOH storing liquid, is maintained at 70 DEG C and stores for future use.

(2) source of iron, PEI and solvent are vacuumized into 5min~30min after mixing, are subsequently filled argon (Ar) gas shielded, 100 DEG C~180 DEG C are heated to, step 1 (1) resulting NaOH storing liquid is added, until the concentration of NaOH is 0.03mol/ in solution L~0.4mol/L is heated to 190 DEG C~230 DEG C after solution colour blackening, continues to be condensed back 30min~2h, reaction knot Beam obtains the reaction solution containing positively charged MNC；

After the reaction solution containing positively charged MNC is cooling, Magneto separate removes reaction solution, by Magneto separate product ultrasound Washing, then Magneto separate, remove reaction solution, and obtaining Magneto separate final product is MNC；MNC is suspended in the distilled water of no RNA enzyme and is made It is standby to obtain MNC solution；

Wherein, the solvent is the mixed liquor of DEG or DEG and ethylene glycol (Ethylene gl ycol, EG), described mixed The volume ratio for closing EG and DEG in liquid is 1:14~1:4, preferably 2:13；

The source of iron is to contain Fe²⁺, the substance stablized under room temperature, can dissolve and not react in the solvent, Preferably FeSO₄·7H₂O or FeCl₂·4H₂O；

The PEI is used as crosslinking agent and stabilizer；

The mass ratio of source of iron and PEI are 1:60~128:60；

It is preferred that vacuumizing 25min after mixing；

160 DEG C are preferably heated to, the resulting NaOH storing liquid of step 1 (1) is added；

It is preferably heated to 220 DEG C to continue to be condensed back 1h, reaction terminates；

It is preferred that first dispersing supersound washing in dehydrated alcohol for Magneto separate product, it is redispersed in ultrasound in deionized water and washes It washs；Supersound washing 3 times repeatedly more preferably in dehydrated alcohol, in deionized water supersound washing 5 times repeatedly；

Can be as needed, different-grain diameter size is prepared by adjusting the time vacuumized or NaOH storing liquid additional amount MNC, such as:

(1) when it is fixed value that NaOH storing liquid volume, which is added, 5min~30min is vacuumized by adjusting, can be prepared Partial size is the MNC of 30nm~196nm；

(2) when evacuated between be fixed value when, NaOH additional amount variation, can be prepared partial size be 30nm~ The MNC of 196nm.

Step 2: being modified with the preparation of the leucocyte film fragment (referred to as M) of nitrine choline

Leucocyte is cultivated into 20h~28h in cell culture complete medium, change contains 0.1mM~0.4mM nitrine gallbladder Alkali (N₃) cell culture complete medium in cultivate 22h~28h, collect leucocyte, be resuspended in buffer solution, use is even Pulp grinder carries out interval to the leucocyte being resuspended in buffer solution and is crushed, broken to interval using sucrose density gradient centrifugation Leucocyte fragment afterwards carries out separation and Extraction, obtains M；By M dissolution, (purchased from match silent winged generation, science and technology is public with Hepes C buffer Department) in, obtain M solution

Wherein, preferably leucocyte is J774A.1 cell line；

It is preferred that at 37 DEG C, CO₂It is cultivated in the constant incubator that concentration is 5%；

It is preferred that being cultivated for 24 hours in cell culture complete medium；

It is preferred that change contains N₃Cell culture complete medium in cultivate for 24 hours；

It is preferred that the cell culture complete medium is to include 10% volume fraction fetal calf serum (FBS), 60 μ g/mL moulds The DMEM complete medium (being purchased from Thermo Fischer Scient Inc.) of element and 100 μ g/mL streptomysins；

Preferably comprise N₃Cell culture complete medium in the N containing 0.1mM₃；

It is preferred that using2 grades of Dispersers and Shakers refiner carry out interval to leucocyte and are crushed.

Step 3: loading the preparation of the biomimetic type magnetic corpusculum (referred to as M-M:siRNA) of siRNA

(1) MNC solution made from step 1 is mixed with siRNA, be vertically suspended 5min~70min at 4 DEG C~8 DEG C, Substance after Magneto separate is resuspended in the PEI aqueous solution of no RNA enzyme by Magneto separate, and the 20min that is vertically suspended at 0 DEG C~8 DEG C~ 60min, Magneto separate obtain MNC:siRNA；MNC:siRNA is dissolved in the distilled water of no RNA enzyme, obtains MNC:siRNA solution；

Wherein, the molar ratio of siRNA and MNC is 1:5~1:15, preferably 1:15；

It is preferred that siRNA is dissolved in the distilled water of no RNA enzyme, obtains siRNA solution and mixed with MNC solution；

It is preferably added to siRNA, be vertically suspended 60min at 4 DEG C；

It is preferred that the concentration of the PEI aqueous solution without RNA enzyme is 0.024g/mL；

It is preferred that substance after Magneto separate is resuspended in the PEI aqueous solution of no RNA enzyme, be vertically suspended 30min at 4 DEG C.

(2) M solution made from MNC:siRNA solution made from step 3 (1), step 2 and buffer are mixed, M is opposite In MNC:siRNA excess, be vertically suspended 6h~15h at 0 DEG C~8 DEG C, and Magneto separate is collected to obtain the MNC of surface package M: SiRNA, i.e. M-MNC:siRNA, for a kind of biomimetic type magnetic corpusculum for loading siRNA of the present invention；

Wherein, be preferably vertically suspended at 4 DEG C 8h；

It in step (1) and (2), is preferably disposed on vertical suspension instrument and is vertically suspended, more preferably carried out with the revolving speed of 20r/min It is vertical to be suspended.

According to different use needs, a kind of biomimetic type magnetic corpusculum loading siRNA of the present invention can be given birth to Object modification obtains the biomimetic type magnetic corpusculum of the loading siRNA of coupled antibody or peptide a kind of: for example according to the need of targets neoplastic cells It wants, by modification group by peptide or antibody modification to a kind of the white thin of the biomimetic type magnetic corpusculum for loading siRNA of the present invention On after birth, steps are as follows:

(1) modification group aqueous solution and antibody or peptide aqueous solution are mixed to get mixed liquor, at room temperature vertical suspension 8h ~12h removes unreacted modification group, antibody or peptide small molecule, be prepared antibody with cycloalkyne modification group or The solution of peptide.

Wherein, the molar ratio of modification group and antibody or peptide is 1:5；

It is preferably disposed on vertical suspension instrument and is vertically suspended, vertical suspension 10h is more preferably carried out with the revolving speed of 20r/min；

Dialysis can be used in unreacted modification group, antibody or peptide small molecule or the method for ultrafiltration removes.

(2) M-MNC:siRNA is dissolved in the distilled water of no RNA enzyme and obtains M-MNC:siRNA solution, and have cycloalkyne The solution mixing of the antibody or peptide of modification group, be vertically suspended 1h~3h at 0 DEG C~8 DEG C, Magneto separate, wherein N is had on M₃ Group passes through N₃With click (click) chemical reaction of antibody or peptide with cycloalkyne modification group, make to repair with cycloalkyne The antibody for adoring group or peptide modification obtain the bionical of the loading siRNA of bio-modification Post functionalization to the surface M-MNC:s iRNA The biomimetic type magnetic corpusculum of the loading siRNA of type magnetic corpusculum, i.e., a kind of coupled antibody or peptide.

Wherein, the molar ratio of M-MNC:siRNA and antibody or peptide with cycloalkyne modification group is 1:5~1:15, excellent It is selected as 1:10；

It is preferred that the 1h that is vertically suspended at 4 DEG C.

It is preferably disposed on vertical suspension instrument and is vertically suspended, be more preferably vertically suspended with the revolving speed of 20r/min.

Specific biological modification method can are as follows:

(1) by diphenyl cyclooctyne-polyethylene glycol (2000)-maleimide (DBCO-PEG (2000)-Mal (maleimide), referred to as DBCO-PEG-Mal) aqueous solution and arginine-glycine-aspartic acid (Arg-Gly-Asp, letter Referred to as RGD) aqueous solution mixing, vertical suspension 8h~12h, then removes unreacted DBCO-PEG-Mal and RGD at room temperature, Obtain connection product (referred to as DBCO-PEG-Mal-RGD) solution of DBCO-PEG-Mal and RGD；

(2) M-MNC:siRNA is dissolved in the distilled water of no RNA enzyme and obtains M-MNC:siRNA solution, with DBCO-PEG- The mixing of Mal-RGD solution, be vertically suspended 1h~3h at 0 DEG C~8 DEG C, Magneto separate, wherein N is had on M₃Group passes through N₃With The click of DBCO-PEG-Mal is chemically reacted, and is made DBCO-PEG-Mal-RGD modification to the surface M-MNC:s iRNA, is obtained biology The biomimetic type magnetic corpusculum (referred to as DBCO-PEG-Mal-RGD-M-MNC:siRNA) of the loading siRNA of Post functionalization is modified, i.e., A kind of biomimetic type magnetic corpusculum of the loading siRNA of coupled antibody or peptide.

Beneficial effect

1. the MNC in the biomimetic type magnetic corpusculum has the present invention provides a kind of biomimetic type magnetic corpusculum for loading siRNA Positive charge, adsorbable siRNA, outside cladding M it is bionical, can efficiently, be rapidly gathered in cell, reach tumor focus position, together When avoid the bodies obstacles such as the immune clearance of organism immune system, the phagocytosis of macrophage, reach the good effect of oncotherapy Fruit；

2. the preparation method is in MNC the present invention provides a kind of preparation method of biomimetic type magnetic corpusculum for loading siRNA In preparation process, using PEI as stabilizer, Fe²⁺Source of iron is reacting precursor, and DEG and/or EG are solvent, in alkaline environment, is utilized The oxidation of remaining oxygen in reaction system solvent, oxidized portion Fe²⁺Generate Fe³⁺, then generated by coprecipitation reaction Fe₃O₄Monocrystalline；In Fe₃O₄Single-crystal surface wraps up PEI and is assembled into MNC by the crosslinked action of PEI；

The MNC uniform particle sizes as made from the preparation method, pumpdown time or NaOH when being prepared by changing add Enter amount, controllable MNC particle size, water-soluble and favorable dispersibility has very strong superparamagnetism, and magnetic moment is with partial size Increase be remarkably reinforced；Solve the problems, such as that existing MNC preparation method operation is complicated, repeatability is poor, expensive, with Meet the fields such as biomedicine to high performance magnetic Fe₃O₄The demand of nano material；

3. the present invention provides a kind of preparation method of biomimetic type magnetic corpusculum for loading siRNA, the preparation method is utilized MNC has positive charge, can be used to adsorb the siRNA with negative electrical charge, loads siRNA method, simply, easy to operate, easily separated； Using PEI by MNC:siRNA surface conversion be positive charge, be coated in M using electrostatic interaction again, it is easy to operate；By In the bionical effect of M, M-M:siRNA can not can escape leucocyte by leukocyte recognition in cyclic process in vivo Phagocytosis extends the circulation time of siRNA in vivo；

4. the method for modifying is white the present invention provides a kind of method of modifying of biomimetic type magnetic corpusculum for loading siRNA The culture medium containing nitrine choline is changed in cell cultivation process, and leucocyte film can successfully be modified to azido group, it is white thin Azido group on after birth can click chemistry mild with DBCO occurrence condition, efficient react；Many functional proteins and peptide fragment can DBCO in modification, therefore the multifunctional protein and peptide fragment can be connected on leucocyte film, then complete the biomimetic type The multifunctional bio of magnetic corpusculum is modified；

5. the present invention provides a kind of method of modifying of biomimetic type magnetic corpusculum for loading siRNA, the method for modifying is made To be connected with the core of the M-MNC:siRNA of antibody or peptide by modification group be the MNC with superparamagnetism, particle size is about 30nm~196nm, can lead to solid tumor high-permeability and delay (EPR) effect and magnetic enrichment effect be gathered in tumor locus；Together Shi Kangti or peptide (RGD) can also the highly expressed α in targets neoplastic cells surface_vβ₃Integrin can also further speed up M-MNC:siRN It is enriched in tumor locus, plays the antitumor action of siRNA drug.

Detailed description of the invention

Fig. 1 is sucrose density gradient centrifugation separation and Extraction epicyte protein result (left side) and protein spots in embodiment 1 Hybridize (Dot Blot) analysis result (right side).

Fig. 2 is transmission electron microscope (TEM) figure of the MNC of different average grain diameters in embodiment 1.

High resolution transmission electron microscopy (HRTEM) figure that Fig. 3 is the MCN that average grain diameter is 81nm in embodiment 1.

The energy spectrum diagram (EDS) that Fig. 4 is the MNC that average grain diameter is 81nm in embodiment 1.

Fig. 5 is X-ray diffraction (XRD) map of the MNC of different average grain diameters in embodiment 1.

Infrared spectroscopy (FTIR) figure that Fig. 6 is the MNC that average grain diameter is 81nm in embodiment 1.

Fig. 7 is the photo on (left side) (right side) afterwards before the separation of average grain diameter is 81nm in embodiment 1 MNC in magnetic field.

Fig. 8 is curve of the magnetization curve of the MNC of different average grain diameters in embodiment 1 after Langewan equation model.

Fig. 9 is the leucocyte film for being modified with nitrine choline that DBCO-Fluor 525 is added in embodiment 1, DBCO- is added The leucocyte film of the unmodified nitrine choline of Fluor 525 and the unmodified nitrine choline that DBCO-Fluor 525 is not added The laser confocal microscope image and flow cytometer fluorescence intensity of leucocyte film characterize curve.

After Figure 10 connect for DBCO-PEG-Mal in embodiment 1 with RGD, the ground substance assistant laser of DBCO-PEG-Mal-RGD Desorption ionization flight time mass spectrum (MALDI-TOF-MS).

Figure 11 is the HRTEM image of M-MNC:sihTERT obtained in embodiment 1.

Figure 12 is M, M-MNC:sihTERT, M obtained in embodiment 1^-And M^-- MNC:sihTERT DBCO-Fluor Laser confocal microscope image after 525 dyeings.

Figure 13 is that agarose gel electrophoresis detects MNC:sihTERT, M-MNC:sihTERT and DBCO-PEG- in embodiment 1 Result of the Mal-RGD-M-MNC:sihTERT to the protective effect of siRNA.

Figure 14 is that leucocyte is detected in embodiment 1 to MNC:sihTETR, M-MNC:sihTERT, R-M-MNC:sihTETR And the laser confocal microscope image of PEG-MNC:sihTERT endocytosis.

Figure 15 is that human breast cancer cell (MCF-7 cell) is detected in embodiment 1 to MNC:sihTETR, M-MNC: The laser confocal microscope of the phagocytosis effect of sihTERT, R-M-MNC:sihTET and R-M-MNC:sihTERT (m+) is imaged Figure.

Figure 16 is to detect sihTERT silencing hTERT albumen with detected by Western blot (Western-blot) in embodiment 1 Effect picture.

Figure 17 is to detect sihTERT using detection cytotoxicity method in embodiment 1 to inhibit tumour cell capability result figure.

Figure 18 is that human breast cancer cell (MB-MDA-231 cell line) is detected in embodiment 3 to MNC:siGFP, M-MNC: The laser confocal microscope image of siGFP, R-M-MNC:siGFP and R-M-MNC:siGFP (m+) phagocytosis effect.

Figure 19 is that MNC:siGFP, M-MNC:siGFP, R-M-MNC:siGFP and R-M-MNC:siGFP are detected in embodiment 3 (m+) the laser confocal microscope image of silences green fluorescent albumen (GFP) ability.

Specific embodiment

Below with reference to embodiment, the present invention is described in detail with attached drawing.

In following embodiment:

The Magneto separate specifically uses method are as follows: uses magnetic suck, reaction solution is removed with liquid-transfering gun, obtain surplus materials As Magneto separate product.

Vertical be suspended is to be placed on vertical suspension instrument to be vertically suspended with the revolving speed of 20r/min.

The distilled water of no RNA enzyme is purchased from Beijing Suo Laibao Science and Technology Ltd；

Amorphous carbon film copper mesh is D200, reaches Zico instrument Science and Technology Ltd. purchased from Beijing；

Hepes B buffer and Hepes C buffer are purchased from Thermo Fischer Scient Inc.；

Protease inhibitors is purchased from Roche Holding Ag；

The cell culture complete medium be include 10% volume fraction fetal calf serum (FBS), 60 μ g/mL penicillin and The DMEM complete medium of 100 μ g/m streptomysins is purchased from Thermo Fischer Scient Inc.；

The dyestuff DBCO-Fluor 525 of cycloalkyne is purchased from Click Chemistry Tools；

SUPER Green II, rhodamine-phalloidine dyestuff and hochest dyestuff are purchased from the general rich biochemistry in Beijing Co., Ltd；

Primary antibody CD45 and the rabbit anti-mouse secondary antibody of horseradish peroxidase-labeled are purchased from the limited public affairs of Ai Bokang (Shanghai) trade Department；

Tris-Buffered Saline Tween-20 (TBST), phosphate buffer (PBS) and polyvinylidene fluoride (PVDF) sheep blood serum that film and mass fraction are 1% is purchased from Beijing Suo Laibao Science and Technology Ltd；

CCK-8 cytoactive detection kit is purchased from the green skies, China；

PCR kit for fluorescence quantitative is purchased from Takara, Japan；

Western-blot kit is purchased from Invent Biotechnologies, the U.S.；

Other agents useful for same are purchased from Sigma Corporation；

Transmission electron microscope (TEM) uses the JEM-2100F of Jeol Ltd. (JEOL)；

Energy disperse spectroscopy (EDS) uses the Aztec of Oxford Instruments；

X-ray diffraction (XRD) instrument uses XPert PRO MPD, PANalytical, Holland；

Infrared spectroscopy (FTIR) instrument uses the FT/IR660 of JASCO company；

Refiner is using IKA company, GermanyDispersers and Shakers refiner,T18；

Centrifuge and centrifuge tube are purchased from Beckman Coulter Inc., the U.S. (Beckman Coulter, Inc.), from Scheming uses Beckman Coulter L-100XP Ultracentrifuge；

Laser confocal microscope comes from UltraVIEW VoX, PerkinElmer, the U.S.；

Flow cytometer comes from Beckman Coulter, cyan；

LCQ DecaXP spectrometer MALDI mass spectrograph comes from Thermo Fisher, the U.S..

Embodiment 1

A kind of preparation method for the biomimetic type magnetic corpusculum loading siRNA, specific step is as follows for the method:

Step 1: the preparation of MNC

(1) by DEG in the environment of Ar gas, after oxygen is removed completely, solid NaOH is dissolved into DEG, is added simultaneously Heat is warming up to 120 DEG C, stops heating when solid NaOH is completely dissolved, and the preparation process whole process all uses Ar gas shielded, makes It is standby to obtain pale yellow solution, it is NaOH storing liquid, is maintained at 70 DEG C and stores for future use；

(2) by FeSO₄·7H₂O, PEI and solvent vacuumize 25min after mixing, fill Ar gas shielded later；Heating 160 DEG C are warming up to, step 1 (1) resulting NaOH storing liquid is added, until the molar concentration of NaOH is 0.03mol/L in solution, Solution colour becomes black after about 3min, then heats to 220 DEG C, continues to be condensed back 1h, reaction terminates, obtains containing band just The reaction solution of the MNC of charge；

After the reaction solution containing positively charged MNC is cooling, Magneto separate removes reaction solution, and Magneto separate product is dispersed The supersound washing in dehydrated alcohol, 3 times repeatedly, Magneto separate disperses Magneto separate product in deionized water, and supersound washing 5 times, Magneto separate final product is obtained, is MNC；MNC is dissolved in the distilled water of no RNA enzyme, obtains MNC solution, concentration is 50 μ g/ mL。

Wherein, solvent is the mixed liquor of DEG and EG, and wherein the volume of EG and DEG is 1:7.

PEI is as crosslinking agent and stabilizer；

FeSO₄·7H₂The mass ratio of O and PEI is 1:60.

Can be as needed, in the case where other preparation conditions are constant, different grains are prepared by adjusting the time vacuumized The MNC of diameter size, as shown in table 1:

Table 1

Or in the case where other preparation conditions are constant, adjust NaOH storing liquid additional amount be 0.4mL, 0.6mL, 0.75mL, 0.8mL, 0.85mL, 1.2mL, 1.5mL and 2mL prepare the MNC of different-grain diameter size, as shown in table 2

Table 2

Step 2: the preparation of M

Leucocyte J774A.1 cell line is placed in 37 DEG C, CO₂It is complete with cell culture in the constant incubator that concentration is 5% Full culture medium culture for 24 hours, changes to and continues culture in the cell culture complete medium of the nitrine choline containing 0.1mM for 24 hours, collect Leucocyte discards culture medium.

The leucocyte being collected into is suspended again with the Hepes B buffer of pH 7.6, the protease suppression of 10 μ L/mL is added Preparation, with 2 grades of interval smudge cells of refiner: broken 2min stops 1min, is repeated 10 times, and then 1000r/min is centrifuged 3min, Supernatant is collected, is precipitated with having added the Hepes B buffer of 10 μ L/mL protease inhibitors to suspend again, with 2 grades of intervals of refiner Smudge cells: broken 2min stops 1min, is repeated 10 times, and then 1000r/min is centrifuged 3min, collects supernatant, and supernatant is equally outstanding Float in the Hepes B buffer containing protease inhibitors, supernatant is mixed into the clasmatosis liquid of last time twice, obtains Broken leucocyte fragment in buffer solution carries out separation and Extraction epicyte protein using sucrose density gradient centrifugation, obtains M is dissolved in Hepes C buffer by M, obtains M solution, and concentration is 20.33 μ g/mL.

The sucrose density gradient centrifugation separation and Extraction epicyte protein specifically: in centrifuge tube, from top to bottom successively The sucrose solution that laying quality score is 55%, 40% and 30%, 4 DEG C of standing 4h；Be added M solution, using centrifuge 4 DEG C, 28000g is centrifuged 2h, as shown in the left side Fig. 1, occurs the band of three gradients in centrifuge tube, being followed successively by mass fraction from bottom to top is 55%, 40% and 30% band collects the band respectively, suspends separation again in quality with Hepes C buffer respectively The protein band in sucrose solution that score is 55%, 40% and 30%, with centrifuge at 4 DEG C, 28000g is centrifuged 30min and carries out Desugar processing, obtains being precipitated as M, carries out protein spots hybridization (Dot blot) analysis to M, method particularly includes: by described three Band each takes 5 μ L samples successively to put on polyvinylidene fluoride (PVDF) film respectively, the sheep for being 1% with mass fraction after air-drying Serum closing overnight, the pvdf membrane closed is dipped in the primary antibody CD45 solution diluted with PBS, the body of CD45 and PBS Product is taken out after concussion incubation 1h, is shaken with Tris-Buffered Saline Tween-20 (TBST) buffer than being 1:200 It washes 3 times, then pvdf membrane is dipped to in the rabbit anti-mouse two corresponding anti-solution of the PBS horseradish peroxidase-labeled diluted, institute The volume ratio for stating secondary antibody and PBS is 1:2000, and concussion is taken out after being incubated for 1h, is washed 3 times with the concussion of TBST buffer, finally by PVDF Film is immersed in concussion in DAB solution and is incubated for 10min~30min, takes out pvdf membrane later, dries after being rinsed with deionized water, The spot on film is observed in gel imager, for analysis result as shown in the right side Fig. 1, there are three spots from bottom to top in pvdf membrane, according to Secondary is the band that mass fraction is 55%, 40% and 30%, it is seen that the band that mass fraction is 40% corresponds to the color of spot most Deep, effect is best, therefore the M of 40% sucrose gradient separation and Extraction is selected to coat MNC.

Step 3: the preparation of M-M:siRNA

(1) sihTERT (hTETR: people for dissolving average grain diameter without RNA enzyme distilled water for addition in the MNC solution of 81nm Telomerase reverse transcriptase gene), the molar ratio of sihTERT and MNC are 1:10；It is placed in vertical suspension instrument, with 20r/ at 4 DEG C Min is vertically suspended 60min, and Magneto separate removes unadsorbed sihTERT, by substance after Magneto separate be resuspended in 0.024g/mL without In the PEI aqueous solution of RNA enzyme, it is placed in vertical suspension instrument, is vertically suspended 60min at 4 DEG C with 20r/min, Magneto separate obtains band The MNC:sihTERT of positive charge is dissolved in the distilled water of no RNA enzyme, obtains MNC:sihTERT solution, and concentration is 50 μ g/ mL。

(2) MNC:sihTERT solution, M solution and Hepes C buffer are mixed, vertical suspension instrument is placed in, at 4 DEG C It is vertically suspended 8h with 20r/min, Magneto separate is collected to obtain MNC:sihTERT, the i.e. M-MNC:sihTERT of surface package M, for this The biomimetic type magnetic corpusculum of invention loading siRNA a kind of.

Bio-modification is carried out to M-MNC:sihTERT, method of modifying is as follows:

(1) DBCO-PEG-Mal aqueous solution is mixed with RGD aqueous solution, wherein the molar ratio of DBCO-PEG-Mal and RGD For 1:5, it is placed in vertical suspension instrument, is vertically suspended 12h with 20r/min at room temperature, dialysis removes unreacted DBCO-PEG- Mal and RGD obtains DBCO-PEG-Mal-RGD solution, concentration 37mM；

(2) the M-MNC:sihTERT distilled water for being dissolved in no RNA enzyme is obtained concentration is 50 μ g/mL M-MNC: SihTERT solution mixes with DBCO-PEG-Mal-RGD solution, wherein M-MNC:sihTERT and DBCO-PEG-Mal-RGD Molar ratio is 1:10, is placed in vertical suspension instrument, is vertically suspended 1h at 4 DEG C with 20r/min, and Magneto separate obtains DBCO-PEG- Mal-RGD-M-MNC:sihTER T (can be abbreviated as R-M-MNC:sihTERT), for a kind of loading after bio-modification The biomimetic type magnetic corpusculum of siRNA,.

Test characterization experiment:

1. it is as follows that pair MNC solution being prepared carries out test characterization experiment:

I, each 10 μ L of MNC solution of different-grain diameter made from difference Example 1, is dispersed in respectively in 1mL deionized water, MNC solution after being diluted, the MNC solution after taking 10 μ L to dilute are dripped respectively in amorphous carbon film copper mesh carrier, after drying, It is observed by transmission electron microscope (JEM-2100F), as shown in Figure 2.Pumpdown time be 30min, 25min, When 20min, 15min and 5min, the MNC of acquisition in Fig. 2 successively respectively as schemed shown in i, figure ii, figure iii, figure iv and figure v, it is known that The average grain diameter of MNC is respectively 31nm, 81nm, 110nm, 140nm and 196nm, meanwhile, the dispersibility of the MNC can be observed Good and partial size is more uniform；The MNC that average grain diameter is 81nm is observed with high resolution transmission electron microscopy, such as Fig. 3 institute Show, the MNC that display average grain diameter is 81nm is not a complete single crystal grain, but is about 11nm's by many partial sizes Fe₃O₄The cluster that monocrystalline is constituted, while can also be observed that external fuzzy PEI layer, the upper right corner in the left box of Fig. 3 and the right side Fig. 3 Illustration be lattice direction enlarged drawing, the spacing by measuring adjacent crystal planes is 0.29nm, Fe with face-centred cubic structure₃O₄ The spacing of lattice of crystal (220) crystal face is consistent.

II, by average grain diameter be 81nm MNC energy spectrometer analysis, as a result as shown in Figure 4, it is seen that its element group become C, O, there is the peak not marked from amorphous carbon film copper mesh carrier in Fe, Fig. 4.

Each 0.5g of MNC of difference average grain diameter made from Example 1 respectively is respectively adopted X-ray diffractometer progress X and penetrates The test of line diffraction uses wavelength to acquire data for the Cu-K alpha ray of 0.15406nm, and 2 θ of the angle of diffraction is 25 °~70 °, obtains corresponding Diffracting spectrum as a result, as shown in Figure 5, it is seen that the curve in Fig. 5 from top to bottom successively indicate average grain diameter be 31nm, 81nm, The MNC diffraction curve of 110nm, 140nm and 196nm, occur in diffraction curve characteristic peak 2 angles θ respectively be 30.4 °, The peak type that the characteristic peak is proved after 35.6 °, 43.4 °, 53.4 °, 57.4 ° and 62.7 °, with the comparison of JCPDS card is Fe₃O₄ The characteristic diffraction peak of crystal, the corresponding Fe of difference₃O₄Crystal face be followed successively by (220), (311), (400), (422), (511) and (440), without miscellaneous peak, diffraction maximum is more sharp, shows that the MNC crystallinity is higher.

III, average grain diameter is vacuum dried for the MNC of 81nm, it takes and is ground on a small quantity with KBr crystal, made after compressing tablet process Sample is obtained, sample is subjected to infrared spectrum analysis using infrared spectrometer, as a result as shown in fig. 6, being in wave number as shown in Figure 6 586.70cm^-1There is absorption peak at place, and peak type is sharp, is Fe-O key characteristic absorption peak；1613.76cm^-1Place is that N-H key is curved in amino Bent vibration absorption peak, 1053.07cm^-1For C-N key stretching vibration absworption peak；Free amine group is in 3500cm^-1~3400cm^-1It answers at place There are two obvious absorption bands, but in 3418.20cm^-1Locate only one wide and strong absorption peak, it was demonstrated that PEI is wrapped in described The surface MNC causes it to change variation and two absorption peaks is caused to become one.

IV, the MNC has very strong magnetic navigability, and taking average partial size is that the MNC of 81nm is dispersed in water, and presents such as Fig. 7 a left side shown in dark solution, by magnet Magneto separate, only about 10s can complete concentration and separation, solution by black become clarify, As shown in the right side Fig. 7；Average grain diameter is that the MNC of 31nm, 110nm, 140nm and 196nm carry out the magnetic navigability experiment, as a result It is similar.

V, (300K) under room temperature, by after the MNC vacuum drying of the different average grain diameters, vibrating specimen magnetometer is carried out (VSM) magnetization curve is obtained after testing, as shown in Figure 8, it is seen that the curve in Fig. 8 in five curve graphs respectively represents average grain diameter For the magnetization curve of the MNC of 31nm, 81nm, 110nm, 140nm and 196nm, corresponding specific saturation magnetization difference is successively For 61.99emu/g, 71.39emu/g, 73.05emu/g, 76.37emu/g and 80.78emu/g, numerical value is with average grain diameter Increase and increases；Simultaneously it can be seen that magnetization curve is all " S " type, remanent magnetism and coercivity are all 0, are illustrated, although MNC under room temperature Partial size shows superparamagnetism in 24.8nm or more.

2. it is as follows that pair M solution being prepared carries out test characterization experiment:

(1) it is reacted using click chemistry (Click), the dyestuff (DBCO-Fluor 525) of cycloalkyne is made to mark leukon The cell membrane of J774A.1 modifies feelings to the nitrine choline of the cell membrane by laser confocal microscope and flow cytometer Condition is characterized, the specific steps are as follows: the leucocyte of J774A.1 cell line is placed in 37 DEG C, CO₂The constant temperature that concentration is 5% is trained It supports in case, for 24 hours with the culture of cell culture complete medium, changes to the cell training of the nitrine choline containing 0.1mM and 0.4mM respectively Feeding complete medium continues culture for 24 hours.

Culture solution is discarded, the fixed 15min of the paraformaldehyde that the volume fraction that 1mL is added is 4% removes paraformaldehyde, uses Phosphate buffer washs 2 times；It is added 10 μM of 525 dyestuff of DBCO-Fluor later, after 37 DEG C of incubation 1h, outwells dyestuff, then It is washed 2 times with PBS, PBS is added and obtains sample solution, carried out laser co-focusing fluorescence imaging and flow cytometer characterization fluorescence is strong Degree detection.

The sample obtained after the cell culture complete medium of the nitrine choline containing 0.1mM continues culture for 24 hours is molten Liquid carries out laser co-focusing fluorescence imaging testing result as shown in c1 in Fig. 9, has red fluorescent, illustrates to have on cell membrane folded Nitrogen choline is simultaneously dyed by 525 dyestuff of DBCO-Fluor；Flow cytometer characterizes fluorescence intensity testing result such as c2 institute in Fig. 9 Show, having fluorescence signal value is 1215.25, and than a2, b2 will be high, shows the azido group on cell membrane by DBCO-Fluor 525 dyestuffs are dyed, while being also turned out and being successfully modified with azido group on J774A.1 cell membrane.

The sample obtained after the cell culture complete medium of the nitrine choline containing 0.4mM continues culture for 24 hours is molten Liquid carries out laser co-focusing fluorescence imaging testing result as shown in d1 in Fig. 9, has red fluorescent, illustrates to have on cell membrane folded Nitrogen choline is simultaneously dyed by 525 dyestuff of DBCO-Fluor；Flow cytometer characterizes fluorescence intensity testing result such as d2 institute in Fig. 9 Show, having fluorescence signal value is 1768.98, and appearance is bimodal, is azido group excessively and is unevenly distributed, illustrates 0.4mM nitrine choline Concentration is higher compared with 0.1mM nitrine choline concentration, in conjunction with DBCO-Fluor 525 it is also more.

(2) control experiment 1: the leucocyte of J774A.1 cell line is placed in 37 DEG C, CO₂The constant incubator that concentration is 5% In, with cell culture complete medium culture 48h.

Culture solution is discarded, the fixed 15min of the paraformaldehyde that the volume fraction that 1mL is added is 4% is washed twice with PBS, carried out Laser co-focusing fluorescence imaging and flow cytometer characterization fluorescence intensity detection.

For laser co-focusing fluorescence imaging testing result as shown in a1 in Fig. 9, redfree fluorescence signal illustrates nothing on cell membrane 525 dyestuff of DBCO-Fluor is not added yet for nitrine choline；Flow cytometer characterizes fluorescence intensity testing result such as a2 institute in Fig. 9 Show, streaming figure fluorescence signal value is 2.59, and signal value is lower.

(3) control experiment 2: the leucocyte of J774A.1 cell line is placed in 37 DEG C, CO₂The constant incubator that concentration is 5% In, with cell culture complete medium culture culture 48h.

Culture solution is discarded, the fixed 15min of the paraformaldehyde that the volume fraction that 1mL is added is 4% is specifically washed twice with PBS； It is added 10 μM of 525 dyestuff of DBCO-Fluor later, after 37 DEG C of incubations 1h, outwells dyestuff, then washed 2 times with PBS, addition PBS Sample solution is obtained, laser co-focusing fluorescence imaging and flow cytometer characterization fluorescence intensity detection are carried out.

Laser co-focusing fluorescence imaging testing result has weaker red fluorescent, illustrates cell as shown in b1 in Fig. 9 Without nitrine choline on film, click chemistry cannot occur with DBCO-Fluor 525 and react, but because DBCO-Fluor 525 is a small amount of non- Specific adsorption is on cell, therefore laser co-focusing fluorescence imaging figure has weaker red fluorescent；Flow cytometer characterization For fluorescence intensity testing result as shown in b2 in Fig. 9, streaming figure streaming figure fluorescence signal value is 94.95, is DBCO-Fluor 525 The non-specific adsorption fluorescent value of dyestuff.

In conclusion can prove that the M being prepared is the leucocyte film fragment for being modified with azido group, selection contains The cell culture complete medium of the nitrine choline of 0.1mM is cultivated.

In Fig. 9, DBCO-Fluor 525 (+), which indicates to be added, DBCO-Fluor 525, and DBCO-Fluor 525 (-) is indicated DBCO-Fluor 525 is not added；Azide-Cho indicates nitrine choline.

3. it is as follows that pair DBCO-PEG-Mal-RGD solution being prepared carries out test characterization experiment:

DBCO-PEG-Mal-RGD solution is taken, is detected on LCQ DecaXP spectrometer MALDI mass spectrograph, is examined The results are shown in Figure 10 for survey, and a shows that DBCO-PEG-Mal connect to form DBCO-PEG-Mal-RGD with RGD, and molecular weight is 1382.025；B is DBCO-PEG-Mal, molecular weight 707.627, c RGD, molecular weight 674.755；Illustrate DBCO-PEG- Mal has been connect with RGD.

4. it is as follows that couple M-MNC:sihTERT being prepared carries out test characterization experiment:

I, M-MNC:sihTERT solution made from 10 μ L embodiments 1 is dispersed in 1mL deionized water and obtains sample, taken About 10 μ L samples drip on the copper mesh of amorphous carbon film, are observed by high resolution transmission electron microscopy, as a result such as Figure 11 It is shown, it is seen that the one layer of slightly transparent leucocyte film in the periphery MNC.

II, it by M and M-MNC:sihTERT 525 dyeing of DBCO-Fluor, is seen under laser confocal microscope It examines.

Check experiment 1: by the leucocyte J774A.1 cell line cell culture complete medium in 1 step 2 of embodiment Culture for 24 hours, continues culture in the cell culture complete medium of nitrine choline of the change containing 0.1mM and is changed to be trained with cell for 24 hours The step of feeding complete medium culture 48h, remaining step is with embodiment 1, the leucocyte film of unmodified nitrine choline can be obtained Fragment (referred to as M^-) solution and M^-- MNC:sihTERT solution.By M^-And M^-- MNC:sihTERT DBCO-Fluor 525 dyeings are observed under laser confocal microscope.

It is as shown in figure 12 that result is observed under laser confocal microscope, scale is 10 μm.Wherein, a1 is M DBCO- Observation after 525 dyeing of Fluor illustrates to be modified with nitrine choline on cell membrane as a result, there is red fluorescence in figure；B1 is M-MNC:sihTERT laser confocal microscope, exciting light 488 emit the reflectogram obtained at light 488-500, show as Green fluorescence illustrates to be modified with nitrine choline on the cell membrane of M-MNC:sihTERT；C1Merge is the stacking chart of a1 and b1, figure In have green and red fluorescence, illustrate to be modified with nitrine choline on the cell membrane of M-MNC:sihTERT.

A2 is M^-With the observation after 525 dyeing of DBCO-Fluor as a result, there is no fluorescence in figure, illustrate on cell membrane Nitrine choline is not decorated, it cannot be by 525 dyeing of DBCO-Fluor；B2 is M^-- MNC:sihTERT laser co-focusing Microscope, exciting light 488 emit the reflectogram obtained at light 488-500, and showing as green fluorescence is M^--MNC:sihTERT； C2Merge is the stacking chart of a2 and b2, there was only green fluorescence in figure, illustrates M^-It is not decorated on the cell membrane of-MNC:sihTERT There is nitrine choline.

III, sihTERT, MNC:sihTERT, M-MNC:sihTERT and DBCO-PEG- are detected with agarose gel electrophoresis Mal-RGD-M-MNC:sihTERT, the specific method is as follows:

SihTERT and DBCO-PEG-Mal-RGD-M-MNC:sihTERT are dissolved in no RNA enzyme double steaming solution respectively It is 5nM sihTERT solution and DBCO-PEG-Mal-RGD-M-MNC:sihTERT solution that concentration, which is made, by 4 kinds of solution point It is not put into the PBS of the FBS containing volume fraction 10% and handles 4h, obtain 4 samples；Control sample is sihTERT solution.It will Respectively at a, b, c of 1% (w/v) Ago-Gel, 3 μ L of point sample at five loading wells of d, e shown for 4 samples and 1 control sample Toner is SUPER Green II, the electrophoresis 30min under 80V voltage.Electrophoresis result is as shown in figure 13, a, b from left to right, c, D and e is followed successively by sihTERT, MNC:sihTERT, M-MNC:sihTERT and DBCO-PEG-Mal-RGD-M- after processing respectively MNC:sihTERT electrophoresis result；For sihTERT by after serum degradation, band is thin out in a, since sihTERT and MNC is adsorbed in b, MNC can reduce degradation of the serum to sihTERT, while limit siRNA ribbon velocity after MNC absorption siRNA, therefore band is thin, And rearward；C and d is respectively M-MNC:sihTERT and DBCO-PEG-Mal-RGD-M-MNC:sihTERT, is occurred without band, Illustrate that M-MNC:sihTERT and DBCO-PEG-Mal-RGD-M-MNC:sihTERT have protective effect to sihTERT, and limits SiRNA electrophoresis.E is the sihTERT of control group, band occurs, illustrates to be not put into and handles in 10% FBS serum SihTETR will not be degraded, the electrophoretic band occurred for normal sihTERT.

5. it is as follows that the tumor suppression ability of couple M-MNC:sihTERT being prepared carries out test characterization experiment:

I, it detects bionical magnetic corpusculum reduction leucocyte and effect is swallowed to it.

The leucocyte of J774A.1 cell line is placed in 37 DEG C, CO₂In the constant incubator that concentration is 5%, cell culture is used Complete medium culture for 24 hours, the MNC:sihTETR that will be walked in embodiment 1, M-MNC:sihTERT, R-M-MNC: SihTETR and the M in step 3 (2) step is changed to PEG, remaining is with embodiment 1, tetra- kinds of obtained PEG-MNC:sihTERT Sample is separately added into J774A.1 cell, is incubated for for 24 hours at 37 DEG C, discards culture medium, and PBS is washed three times, uses rhodamine-ghost respectively Cyclic peptide dyes cell membrane, Hochest dyes nucleus, with confocal laser scanning microscope leucocyte to four kinds of samples Phagocytic activity.As shown in figure 14, red represents cell membrane in figure, and blue represents nucleus, and green is each sample of cell endocytic, As seen from the figure, the M-MNC:sihTERT and R-M-MNC:sihTERT for coating leucocyte film can escape the phagocytosis of leucocyte, cell In green fluorescence be less than MNC:sihTERT and PEG-MNC:sihTERT group.

II, phagocytosis effect of detection human breast cancer cell (MCF-7 cell) to bionical magnetic corpusculum.

Human breast cancer cell (MCF-7 cell) cell line is placed in 37 DEG C, CO₂In the constant incubator that concentration is 5%, use The culture of cell culture complete medium for 24 hours, obtains cell culture fluid, the MNC:sihTETR that embodiment 1 is obtained, M-MNC: SihTERT and R-M-MNC:sihTET are separately added into cell culture fluid, and wherein R-M-MNC:sihTERT group is divided into two parts addition Cell culture fluid respectively plus magnetic group (R-M-MNC:sihTERT (m+)) and is not added magnetic group (R-M-MNC:sihTERT), four groups Sample continues culture for 24 hours in MCF-7 cultivating system, emigrated cells culture solution, dyes in J774A.1 in cell dyeing same 5 I, Cell membrane, nucleus, color sample is the same as 5 I.

As shown in figure 15, MCF-7 cell phagocytosis R-M-MNC:sihTERT (m+) group and R-M-MNC:sihTERT group are more, Green fluorescence in cell is more than MNC:sihTERT and M-MNC:sihTERT group.

III, inhibit tumour cell capacity experimental

Human breast cancer cell (MCF-7 cell) cell line is placed in 37 DEG C, CO₂In the constant incubator that concentration is 5%, with thin Born of the same parents cultivate complete medium culture for 24 hours.MNC:sihTETR, M-MNC:sihTERT and the R- that sihTERT, embodiment 1 are obtained M-MNC:sihTETR is separately added into cell culture fluid, and wherein R-M-MNC:sihTERT group is divided into two parts addition cell culture Liquid respectively plus magnetic group (R-M-MNC:sihTERT (m+)) and is not added magnetic group (R-M-MNC:sihTERT), and five groups of samples exist Continue culture in MCF-7 cultivating system for 24 hours, collects cell；By PCR kit for fluorescence quantitative (Takara, Japan) method detection The ability of sihTETR silencing human telomerase reverse transcriptase (hTERT) gene mRNA that bionical magnetic corpusculum is loaded；Use Western- Blot kit (Invent Biotechnologies, the U.S.) detects sihTETR silencing hTERT protein expression ability, as a result As shown in figure 16.

As seen from Figure 16 load sihTERT's and with RGD peptide bionical magnetic corpusculum group (R-M-MNC:sihTERT) group with It adds magnetic group (R-M-MNC:sihTERT (m+)) to be compared to sihTERT, MNC:sihTERT and M-MNC:siTERT silencing The hTETR potency of gene is more preferable, more to reduce hTERT gene expression protein content, it is seen that sihTERT:R-M-MNC has preferably heavy The effect of silent tumour sihTERT gene.

Inhibit tumour cell ability, knot with CCK-8 cytoactive detection kit (the green skies, China) detection sihTERT Fruit is as shown in figure 17, loads sihTERT's and bionical magnetic corpusculum group (R-M-MNC:sihTERT) group with RGD peptide adds with it Magnetic group (R-M-MNC:sihTERT (m+)) is compared to sihTERT, MNC:sihTERT and M-MNC:siTERT to the poison of cell Property effect it is bigger, can more effectively inhibit growth of tumour cell.

Embodiment 2

Step 1: the preparation of MNC

(1) by DEG in the environment of Ar gas, after oxygen is removed completely, solid NaOH is dissolved into DEG, is added simultaneously Heat is warming up to 100 DEG C, stops heating when solid NaOH is completely dissolved, and the preparation process whole process all uses Ar gas shielded, makes It is standby to obtain pale yellow solution, it is NaOH storing liquid, is maintained at 70 DEG C and stores for future use；

(2) by FeCl₂·4H₂O, PEI and solvent vacuumize 25min after mixing, fill Ar gas shielded later；Heating 100 DEG C are warming up to, step 1 (1) resulting NaOH storing liquid is added, until the molar concentration of NaOH is 0.03mol/L in solution, Solution colour becomes black after about 3min, then heats to 190 DEG C, continues to be condensed back 30min, reaction terminates, contained The reaction solution of positively charged MNC；

Wherein, solvent DEG；

PEI is as crosslinking agent and stabilizer；

FeCl₂·4H₂The mass ratio of O and PEI is 128:60.

Step 2: the preparation of M

Leucocyte J774A.1 cell line is placed in 37 DEG C, CO₂It is complete with cell culture in the constant incubator that concentration is 5% Full culture medium culture 20h is changed to and is continued to cultivate 22h in the cell culture complete medium of the nitrine choline containing 0.4mM, collects Leucocyte discards culture medium.

The leucocyte being collected into is suspended again with the Hepes B buffer of pH 7.6, the protease suppression of 10 μ L/mL is added Preparation, with 2 grades of interval smudge cells of refiner: broken 2min stops 1min, is repeated 10 times, and then 1000r/min is centrifuged 3min, Supernatant is collected, is precipitated with having added the Hepes B buffer of 10 μ L/mL protease inhibitors to suspend again, with 2 grades of intervals of refiner Smudge cells: broken 2min stops 1min, is repeated 10 times, and then 1000r/min is centrifuged 3min, collects supernatant, and supernatant is equally outstanding Float in the Hepes B buffer containing protease inhibitors, supernatant is mixed into the clasmatosis liquid of last time twice, obtains Broken leucocyte fragment in buffer solution carries out separation and Extraction using sucrose density gradient centrifugation, obtains M, M is dissolved in Hepes C buffer, obtains M solution, and concentration is 20.33 μ g/mL.

The sucrose density gradient centrifugation is specifically the same as embodiment 1.

Step 3: the preparation of M-M:siRNA

(1) by average grain diameter to be added in the sihTERT dissolved without RNA enzyme distilled water in the MNC solution of 81nm, The molar ratio of sihTERT and MNC is 1:5；It is placed in vertical suspension instrument, is vertically suspended 60min at 8 DEG C with 20r/min, magnetic point From removing unadsorbed sihTERT, substance after Magneto separate be resuspended in 0.024g/mL without in the PEI aqueous solution of RNA enzyme, is set In vertical suspension instrument, vertically it is suspended 20min at 0 DEG C with 20r/min, Magneto separate obtains positively charged MNC:sihTERT, molten In the distilled water of Xie Yuwu RNA enzyme, MNC:sihTERT solution is obtained, concentration is 50 μ g/mL.

(2) MNC:sihTERT solution, M solution and Hepes C buffer are mixed, vertical suspension instrument is placed in, at 0 DEG C It is vertically suspended 6h with 20r/min, Magneto separate is collected to obtain MNC:sihTERT, the i.e. M-MNC:sihTERT of surface package M, for this The biomimetic type magnetic corpusculum of invention loading siRNA a kind of.

(1) DBCO-PEG-Mal aqueous solution is mixed with RGD aqueous solution, wherein the molar ratio of DBCO-PEG-Mal and RGD For 1:5, it is placed in vertical suspension instrument, is vertically suspended 8h with 20r/min at room temperature, dialysis removes unreacted DBCO-PEG-Mal And RGD, obtain DBCO-PEG-Mal-RGD solution, concentration 37mM；

(2) the M-MNC:sihTERT distilled water for being dissolved in no RNA enzyme is obtained concentration is 50 μ g/mL M-MNC: SihTERT solution mixes with DBCO-PEG-Mal-RGD solution, wherein M-MNC:sihTERT and DBCO-PEG-Mal-RGD Molar ratio is 1:5, is placed in vertical suspension instrument, is vertically suspended 1h at 0 DEG C with 20r/min, and Magneto separate obtains DBCO-PEG- Mal-RGD-M-MNC:sihTER T (can be abbreviated as R-M-MNC:sihTERT), for a kind of loading after bio-modification The biomimetic type magnetic corpusculum of siRNA.

Test characterization experimental method is with embodiment 1, and test sample is to implement 2 to be made, and test result and the test of embodiment 1 are tied Fruit is similar.

Embodiment 3

Step 1: the preparation of MNC

(1) by DEG in the environment of Ar gas, after oxygen is removed completely, solid NaOH is dissolved into DEG, is added simultaneously Heat is warming up to 150 DEG C, stops heating when solid NaOH is completely dissolved, and the preparation process whole process all uses Ar gas shielded, makes It is standby to obtain pale yellow solution, it is NaOH storing liquid, is maintained at 70 DEG C and stores for future use；

(2) by FeSO₄·7H₂O, PEI and solvent vacuumize 25min after mixing, fill Ar gas shielded later；Heating 180 DEG C are warming up to, step 1 (1) resulting NaOH storing liquid is added, until the molar concentration of NaOH is 0.4mol/L in solution, about Solution colour becomes black after 3min, then heats to 230 DEG C, continues to be condensed back 2h, reaction terminates, obtains containing positively charged The reaction solution of the MNC of lotus；

Wherein, solvent is the mixed liquor of DEG and EG, wherein the volume ratio of EG and DEG is 1:4；

PEI is as crosslinking agent and stabilizer；

FeSO₄·7H₂The mass ratio of O and PEI is 128:60.

Step 2: the preparation of M

Leucocyte J774A.1 cell line is placed in 37 DEG C, CO₂It is complete with cell culture in the constant incubator that concentration is 5% Full culture medium culture 28h is changed to and is continued to cultivate 28h in the cell culture complete medium of the nitrine choline containing 0.4mM, collects Leucocyte discards culture medium.

Step 3: the preparation of M-M:siRNA

(1) siGFP that dissolve without RNA enzyme distilled water will be added in MNC solution that average grain diameter is 81nm (GFP is green Fluorescence protein gene) in, the molar ratio of siGFP and MNC are 1:15；It is placed in vertical suspension instrument, it is vertical with 20r/min at 8 DEG C Suspension 70min, Magneto separate remove unadsorbed siGFP, and substance after Magneto separate is resuspended in 0.024g/mL without RNA enzyme In PEI aqueous solution, it is placed in vertical suspension instrument, is vertically suspended 30min at 0 DEG C with 20r/min, Magneto separate obtains positively charged MNC:siGFP is dissolved in the distilled water of no RNA enzyme, obtains MNC:siGFP solution, and concentration is 50 μ g/mL.

(2) by MNC:siGFP solution, M solution and Hepes C buffer mix, be placed in vertical suspension instrument, at 8 DEG C with 20r/min is vertically suspended 15h, and Magneto separate is collected to obtain MNC:siGFP, the i.e. M-MNC:siGFP of surface package M, for the present invention A kind of biomimetic type magnetic corpusculum of the loading siRNA.

Bio-modification is carried out to M-MNC:siGFP, method of modifying is as follows:

(1) with 1 biological modification method step (1) of embodiment；

(2) the M-MNC:siGFP distilled water for being dissolved in no RNA enzyme is obtained concentration is that 50 μ g/mL M-MNC:siGFP are molten Liquid is mixed with DBCO-PEG-Mal-RGD solution, and wherein the molar ratio of M-MNC:siGFP and DBCO-PEG-Mal-RGD is 1: 15, it is placed in vertical suspension instrument, is vertically suspended 3h at 8 DEG C with 20r/min, Magneto separate obtains DBCO-PEG-Mal-RGD-M- MNC:siGFP (can be abbreviated as R-M-MNC:siGFP), and the biomimetic type magnetic for a kind of loading siRNA after bio-modification is small Body.

Test characterization experiment:

Test characterization experimental method 1~4 and 5 I is with embodiment 1, and test sample is to implement 3 to be made, test result and reality It is similar to apply 1 test result of example.

5. it is as follows that the tumor suppression ability of couple M-MNC:siGFP being prepared carries out test characterization experiment:

II, phagocytosis effect of detection human breast cancer cell (MB-MDA-231) to bionical magnetic corpusculum.

Human breast cancer cell (MB-MDA-231) cell line is placed in 37 DEG C, CO₂In the constant incubator that concentration is 5%, For 24 hours with the culture of cell culture complete medium, cell culture fluid is obtained, the MNC:siGFP that embodiment 3 is obtained, M-MNC: SiGFP and R-M-MNC:siGFP are separately added into cell culture fluid, and wherein R-M-MNC:siGFP group is divided into two parts addition cell Culture solution respectively plus magnetic group (R-M-MNC:siGFP (m+)) and is not added magnetic group (R-M-MNC:siGFP), and four groups of samples are in MB- Continue culture in MDA-231 cultivating system for 24 hours, PBS is added in emigrated cells culture solution.

It is observed under laser confocal microscope, as a result as shown in figure 18, green represents cell, and purple is cell endocytic Each sample；MB-MDA-231 cell swallows R-M-MNC:siGFP (m+) group and R-M-MNC:siGFP group is more, is more than MNC: SihTERT and M-MNC:sihTERT group.

III, inhibit tumour cell capacity experimental

Human breast cancer cell (MB-MDA-231) cell line is placed in 37 DEG C, CO₂In the constant incubator that concentration is 5%, use The culture of cell culture complete medium is for 24 hours.MNC:siGFP, M-MNC:siGFP and the R-M- that siGFP, embodiment 3 are obtained MNC:siGFP is separately added into cell culture fluid, and wherein R-M-MNC:siGFP group is divided into two parts addition cell culture fluid, respectively To add magnetic group (R-M-MNC:siGFP (m+)) and being not added magnetic group (R-M-MNC:siGFP), five groups of samples are cultivated in MB-MDA-231 Continue culture in system for 24 hours, is observed under laser confocal microscope, as a result as shown in figure 19.R-M-MNC:siGFP group and R- M-MNC:siGFP (m+), after silences green fluorescent albumen, the green shown as in cell is less, is compared to siGFP, MNC: SiGFP the and M-MNC:siGFP silencing GFP potency of gene is more preferable, it is seen that R-M-MNC:siGFP (m+) has better silencing tumour The effect of siGFP gene.

Claims

1. a kind of biomimetic type magnetic corpusculum for loading siRNA, it is characterised in that: the biomimetic type magnetic corpusculum is referred to as M-MNC: SiRNA, with water-soluble Fe₃O₄Nanocluster is core, outer cladding leucocyte film, in the Fe₃O₄Nanocluster and leucocyte film Between be mounted with siRNA；The Fe₃O₄Nanocluster is by Fe₃O₄Nano crystal and polyethyleneimine composition, positively charged, partial size For 30nm~196nm.

2. a kind of biomimetic type magnetic corpusculum for loading siRNA according to claim 1, it is characterised in that: the Fe₃O₄Nanometer The partial size of cluster is 81nm；Leucocyte film is the cell membrane of leukon J774A.1.

3. the biomimetic type magnetic corpusculum of the loading siRNA of coupled antibody or peptide a kind of, it is characterised in that: as claimed in claim 1 or 2 The outer covalent coupling of leucocyte film of biomimetic type magnetic corpusculum of loading siRNA have antibody or peptide with modification group；

The antibody is the antibody that the end Fc has lysine residue；

The modification group is DBCO-PEG_n- NHS, n=1~2000；

4. the biomimetic type magnetic corpusculum of the loading siRNA of a kind of coupled antibody according to claim 3 or peptide, feature exist In: n=4.

5. a kind of preparation method of the biomimetic type magnetic corpusculum as claimed in claim 1 or 2 for loading siRNA, it is characterised in that: step It is rapid as follows:

Step 1: Fe₃O₄Nanocluster, the i.e. preparation of MNC

(1) under oxygen-free environment, NaOH is completely dissolved in DEG, NaOH storing liquid is prepared；

(2) source of iron, PEI and solvent are vacuumized into 5min~30min after mixing, filling argon gas protection, be heated to 100 DEG C~ 180 DEG C, NaOH storing liquid is added, until the concentration of NaOH is 0.03mol/L~0.4mol/L in solution, after solution colour blackening 190 DEG C~230 DEG C are heated to, continues to be condensed back 30min~2h, obtains MNC reaction solution；

After MNC reaction solution is cooling, Magneto separate removes reaction solution, and Magneto separate product supersound washing, then Magneto separate are removed anti- Liquid is answered, obtaining Magneto separate final product is MNC；MNC is suspended in the distilled water of no RNA enzyme and obtains MNC solution；

The solvent is the mixed liquor of DEG or DEG and EG, and the volume ratio of EG and DEG is 1:14~1:4 in mixed liquor；

The source of iron is to contain Fe²⁺, the substance stablizing under room temperature, can dissolve and not react in the solvent；

The mass ratio of source of iron and PEI are 1:60~128:60；

Step 2: being modified with the leucocyte film fragment of nitrine choline, the i.e. preparation of M

Leucocyte is cultivated into 20h~28h in cell culture complete medium, change contains 0.1mM~0.4mM nitrine choline 22h~28h is cultivated in cell culture complete medium, is collected leucocyte, is resuspended in buffer solution, with refiner to institute It states leucocyte be intermittently crushed, using sucrose density gradient centrifugation to the broken leucocyte fragment separation and Extraction of interval, obtain To M；M is dissolved in Hepes C buffer, M solution is obtained；

Step 3: loading the biomimetic type magnetic corpusculum of siRNA, the i.e. preparation of M-M:siRNA

(1) MNC solution is mixed with siRNA, be vertically suspended 5min~70min at 4 DEG C~8 DEG C, Magneto separate, after Magneto separate Substance is resuspended in the PEI aqueous solution of no RNA enzyme, and be vertically suspended 20min~60min at 0 DEG C~8 DEG C, and Magneto separate obtains MNC:siRNA；MNC:siRNA is dissolved in the distilled water of no RNA enzyme, obtains MNC:siRNA solution；

The molar ratio of siRNA and MNC is 1:5~1:15；

(2) MNC:siRNA solution, M solution and buffer are mixed, be vertically suspended 6h~15h at 0 DEG C~8 DEG C, and Magneto separate is received Collection obtains M-MNC:siRNA, for a kind of biomimetic type magnetic corpusculum for loading siRNA.

6. a kind of preparation method of biomimetic type magnetic corpusculum for loading siRNA according to claim 5, it is characterised in that: step In rapid one:

(1) oxygen-free environment is realized by using inert gas deoxidation treatment and protection；

Being heated to 100 DEG C~150 DEG C is completely dissolved in NaOH in DEG, stops heating when NaOH is completely dissolved；

(2) volume ratio of EG and DEG is 2:13 in DEG and EG mixed liquor；

Source of iron is FeSO₄·7H₂O or FeCl₂·4H₂O；

25min is vacuumized after mixing；

160 DEG C are heated to, NaOH storing liquid is added；

220 DEG C are heated to continue to be condensed back 1h；

Supersound washing in dehydrated alcohol is first dispersed by Magneto separate product, is redispersed in supersound washing in deionized water；

In step 2:

Leucocyte is J774A.1 cell line；

At 37 DEG C, CO₂It is cultivated in the constant incubator that concentration is 5%；

It is cultivated for 24 hours in cell culture complete medium；

Change contains N₃Cell culture complete medium in cultivate for 24 hours；

Cell culture complete medium is to include 10% volume fraction fetal calf serum, 60 μ g/mL penicillin and 100 μ g/mL strepto-s The DMEM complete medium of element；

Contain N₃Cell culture complete medium in the N containing 0.1mM₃；

With2 grades of Dispersers and Shakers refiner carry out interval to leucocyte and are crushed；

In step 3:

(1) molar ratio of siRNA and MNC is 1:15；

SiRNA is dissolved in the distilled water of no RNA enzyme, is obtained siRNA solution and is mixed with MNC solution；

SiRNA is added, be vertically suspended 60min at 4 DEG C；

The concentration of the PEI aqueous solution of no RNA enzyme is 0.024g/mL；

Substance after Magneto separate is resuspended in the PEI aqueous solution of no RNA enzyme, be vertically suspended 30min at 4 DEG C；

(2) 8h that is vertically suspended at 4 DEG C is placed on vertical suspension instrument.

7. a kind of preparation method of biomimetic type magnetic corpusculum for loading siRNA according to claim 6, it is characterised in that: step In rapid one:

(1) DEG is done into deoxidation treatment in the environment of inert gas, after oxygen is removed completely, NaOH is dissolved into DEG, It heats simultaneously, is warming up to 120 DEG C, stop heating when NaOH is completely dissolved, the preparation process whole process all needs inert gas Protection, obtains NaOH storing liquid；

(2) supersound washing 3 times repeatedly in dehydrated alcohol, in deionized water supersound washing 5 times repeatedly；

In step 3:

(2) it is vertically suspended with the revolving speed of 20r/min.

8. a kind of preparation side of the biomimetic type magnetic corpusculum of the loading siRNA of coupled antibody or peptide as described in claim 3 or 4 Method, it is characterised in that: steps are as follows:

(1) modification group aqueous solution and antibody or peptide aqueous solution are mixed to get mixed liquor, at room temperature vertical suspension 8h~ 12h removes unreacted modification group, antibody or peptide small molecule, and antibody or peptide with cycloalkyne modification group is prepared Solution,

The molar ratio of modification group and antibody or peptide is 1:5；

(2) M-MNC:siRNA as claimed in claim 1 or 2 is dissolved in the distilled water of no RNA enzyme and obtains M-MNC:siRNA Solution, and the solution mixing of the antibody with cycloalkyne modification group or peptide, be vertically suspended 1h~3h at 0 DEG C~8 DEG C, magnetic point From, the biomimetic type magnetic corpusculum of the loading siRNA of coupled antibody or peptide is obtained,

The molar ratio of M-MNC:siRNA and antibody or peptide with cycloalkyne modification group is 1:10.

9. the preparation method of the biomimetic type magnetic corpusculum of the loading siRNA of a kind of coupled antibody according to claim 8 or peptide, It is characterized by: steps are as follows:

(1) it is placed on vertical suspension instrument and vertical suspension 10h is carried out with the revolving speed of 20r/min；

Unreacted modification group, antibody or peptide small molecule are removed using the method for dialysis or ultrafiltration；

(2) molar ratio of M-MNC:siRNA and antibody or peptide with cycloalkyne modification group is 1:10；

It is placed at 4 DEG C on vertical suspension instrument and is vertically suspended 1h with the revolving speed of 20r/min.

10. the preparation side of the biomimetic type magnetic corpusculum of the loading siRNA of a kind of coupled antibody according to claim 8 or peptide Method, it is characterised in that:

(1) DBCO-PEG-Mal aqueous solution is mixed with RGD aqueous solution, at room temperature vertical suspension 8h~12h, is removed unreacted DBCO-PEG-Mal and RGD obtains DBCO-PEG-Mal-RGD solution；

(2) M-MNC:siRNA is dissolved in the distilled water of no RNA enzyme and obtains M-MNC:siRNA solution, with DBCO-PEG-Mal- The mixing of RGD solution, be vertically suspended 1h~3h at 0 DEG C~8 DEG C, Magneto separate, obtains the imitative of the loading siRNA of coupled antibody or peptide Raw type magnetic corpusculum.