CN102961337B - Preparation method of target compound nano particle - Google Patents

Preparation method of target compound nano particle Download PDF

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CN102961337B
CN102961337B CN2012105435903A CN201210543590A CN102961337B CN 102961337 B CN102961337 B CN 102961337B CN 2012105435903 A CN2012105435903 A CN 2012105435903A CN 201210543590 A CN201210543590 A CN 201210543590A CN 102961337 B CN102961337 B CN 102961337B
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CN102961337A (en
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郭喜明
郭斌
薄璇
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Harbin Institute of Technology
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Abstract

The invention provides a preparation method of a target compound nano particle, relating to a preparation method of target treatment integration compound nano particles. The invention solves the technical problems of poor carrier stability and small drug-loading quantity of the traditional antineoplastic drugs. The method comprises the following steps of: preparing a methanol solution of an integrated nano particle of a Fe3O4-HCPT@SiO2 coupling porphyrin photosensitizer; and stirring the methanol solution of the integrated nano particle of the Fe3O4-HCPT@SiO2 coupling porphyrin photosensitizer, N,N-dimethylformamide and target biomolecule or antitumor drug for reacting at the room temperature, and centrifuging to obtain the target compound nano particle. Polystyrolsulfon acid (PSS) and 10-hydroxycamptothecine are absorbed on a Fe3O4 magnetic nano particle through a self-assembly mode. The preparation method is high feasibility; the target compound nano particle has a physical target function of the magnetic nano particle and an active target function of further functionally modifying the biomolecule on the surface; and the medicine concentration can be increased under a dual-target function.

Description

The preparation method of targeting composite nanoparticle
Technical field
The present invention relates to the preparation method of the integrated composite nanoparticle of a kind of targeting diagnosis and treatment.
Background technology
According to the official of World Health Organization (WHO), point out: by 2010, malignant tumor will replace cardiovascular disease became the disease that world's death toll is maximum.The clinical diagnosing tumour that can be used for has at present: optical imagery, positron emission tomography, Magnetic Resonance Imaging, ultrasonic and Computed tomography; The treatment tumor has: operation, radiotherapy, the large main method of Drug therapy three, and the householder methods such as photodynamic therapy, thermotherapy, above various therapies have all been received good treatment effect.If with porphyrins, carried out optical imagery, by the stronger absorbing wavelength of its structure porphin ring, be to excite after the 405nm light absorption to produce 630nm and 690nm near infrared region fluorescence to the tumor tissues imaging, have the advantage of low-energy radiation, hypersensitivity etc.; With hydroxy camptothecin (HCPT) class medicine, by selectivity inhibition tumor cell DNA topoisomerase, cut off the DNA single chain, disturb copying of DNA chain, have stronger anti-tumor activity; With porphyrins, carry out optical dynamic therapy, by optionally enrichment on tumor cell of porphyrin, near when using the rayed tumor area of certain wavelength, bringing out biological tissue, the generation activated singlet oxygen of tool (O2) causes tumor cell because of the death of oxidation inactivation, have that antitumor spectra is wide, indication is wide, the remarkable advantage of repetitive therapy repeatedly, and with chemotherapy combined use concertedness good, with the excellent oncotherapy effect of operation associating Reduction surgery scope and minimizing postoperative recurrence; And carry out photo-thermal therapy with golden core/shell nanoparticles, and by the near-infrared wavelength of its adjustable surface longitudinal plasma absorption with its resonance, kill tumor stem cell after release heat, thus suppress neoplasm metastasis; When with the Drug therapy combined effect, have the permeability that the chemical sensitization effect can improve the blood circulation of tumor periphery and strengthen cell membrane, strengthen the inside tumor that penetrates into of medicine.
But in the diagnosing tumor medicine, exist three totally two with problem are: one, target-oriented drug is poor; Hydroxy camptothecin (HCPT) the class medicine of take is example, there are two kinds of forms in HCPT under different pH conditions: Alpha-hydroxy-delta-lactone loop type and carboxylic acid, ethyl ester form, but in two kinds of forms only the lactone structure form have anti-tumor activity, and the water solublity of lactone structure form is poor, how improves lactone structure camptothecine target tumor cell and be the Research Challenges of this type of medicine always; Two,, due to the distinctive biotic environment of tumor tissues, make to reach valid density in diagnosis and treatment drug level difficulty; Three, in the diagnosis and treatment of tumor, use the monotherapy curative effect low, can't treat malignant tumor.Therefore two key problems are that the pharmaceutical carrier that How to choose is suitable improves various Therapeutic Method itself and builds can science and the multiple therapy Comprehensive Treatment of reasonable combination tumour medicine in current tumor diagnosis and treatment research.
Along with scientific and technical development, take micro-nano manufacturing technology and nano material provides an effective way for solving this two large problems as basic diagnosing tumor treatment technology.As after carrier, can make drug targeting act on tumor cell nanoparticle, rear gathering focus, and can strengthen the osmosis of cell to its medicine.The nano-carrier be developed out at present has polymer microballoon nanoparticle, microcapsule lipid and microemulsion, golden nanometer particle etc.; In many nanoparticles, golden nanometer particle has advantages of can be further surface-functionalized tumor cell be had to the biomolecule of targeting such as monoclonal antibody, folic acid, albumen, saccharide etc., after modification, can make nanoparticle accurately be positioned specific tumor cell, significantly improve the concentration of medicine at the specific tumors tissue, further reduce side effect; And golden nanometer particle also can be prepared into and multiplely under specific part and condition, discharge controlled drug delivery system, under the prerequisite that does not weaken the diagnosis and treatment curative effect, reduce simultaneously the consumption of medicine, thereby golden nanometer particle is the desirable nano-carrier of a class.
In recent years on above-mentioned nano-carrier Research foundation, built multiple targeting ability, and the integrated composite Nano of multiple diagnosis and treatment method combination can have been become to a new study hotspot, as based on the liposome nano material with Fe 3O 4The magnetic targeting optical imagery composite nanoparticle that magnetic nano-particle and fluorescent dye build; Based on optical imagery and nuclear magnetic resonance composite nanoparticle, photo-thermal therapy and conventional medicament that golden nanometer particle builds, treat composite nanoparticle.But the research of this type of composite nanoparticle is in the junior stage, still have the defect of three aspects:, first aspect is that the drug loading of this type of drug-loading system is less, stability and targeting lower; Second aspect be in preparation process, need with an organic solvent, surfactant, ultrasonic or homogeneity, this all will limit the diagnosis and treatment process of tumour medicine; The third aspect is difficult to organically combine multiple diagnosis and treatment mode, has long consultation hours.
Summary of the invention
The present invention, in order to solve current antineoplastic drug carrier poor stability, technical problem that drug loading is little, provides a kind of preparation method of targeting composite nanoparticle.
The preparation method of targeting composite nanoparticle is carried out according to following steps:
One, prepare magnetic nano-particle:
By 10mL concentration, be the FeCl of 1mol/L 3Aqueous solution, 2.5mLFeSO 47H 2The HCl solution of O joins the NH that 125mL concentration is 0.7mol/L 37H 2O, continue to stir 30min, and centrifugalize is also collected the product in supernatant, by deionized water wash 3 times of the product in supernatant, and then is dispersed in the 150mL deionized water, obtains Fe 3O 4Nano-particle solution;
Described FeSO 47H 2FeSO in the HCl solution of O 47H 2The concentration of O is that the concentration of 2mol/L, HCl is 2mol/L;
Two, preparation Fe 3O 4-HCPT nanoparticle:
By 10mLFe 3O 4Nano-particle solution is distributed in the NaCl aqueous solution of the poly-hydrogen chloropropene amine of 10ml, ultrasonic reaction 30min, and magnetic separation, be then the NaCl solution washing of 0.51mol/L by concentration, finally is dispersed in water, obtaining concentration is the Fe of the PAH-parcel of 7.5mg/ml 3O 4Nano-particle solution;
In the NaCl aqueous solution of the poly-hydrogen chloropropene amine described in step 2, poly-hydrogen chloropropene amine concentration is 1mg/ml, and the concentration of NaCl is 0.5mol/L; Three, by the Fe of 10mLPAH-parcel 3O 4Nano-particle solution is distributed to ultrasonic reaction 30min in the NaCl aqueous solution of 10ml polystyrolsulfon acid acid sodium, obtains Fe 3O 4The magnetic nano-particle precursor;
In the NaCl aqueous solution of the polystyrolsulfon acid acid sodium described in step 3, the concentration of kayexalate is 1mg/ml, and the concentration of NaCl is 0.5mol/L;
Four, the 364mg10-hydroxy camptothecin is dissolved in 150mLNaOH solution, regulating pH value is 11, then add concentration be the HCl of 1mol/L to regulate pH value be 4.8, obtain the HCPT microemulsion, by 10mlFe 3O 4The magnetic nano-particle precursor joins in the 10mLHCPT microemulsion, stir and pass through the uv absorption monitoring reaction, when the characteristic absorption peak of 10-hydroxycamptothecine disappears, stopped reaction, the magnetic separation collecting precipitation, and with pH value, be 4.8 HCl solution washing, then add in the 150mL deionized water, obtain the Fe of kayexalate/HCPT 3O 4The magnetic nano-particle aqueous solution;
Five, preparation Fe 3O 4-HCPT@SiO 2:
The 1mL ethyl orthosilicate is mixed with 30mL ethanol, obtain the alcoholic solution of ethyl orthosilicate, by 2.5mLNH 3H 2O, 38mLH 2The Fe of O, 38mL ethanol and 10mL kayexalate/HCPT 3O 4The magnetic nano-particle aqueous solution, then add the alcoholic solution of ethyl orthosilicate, reacted 4 hours, and magnetic separation, collecting precipitation, and the mixed solution of water and ethanol washing 3 times, redispersion, in the 100mL deionized water, obtains Fe 3O 4-HCPT@SiO 2Aqueous solution;
Six, at rotating speed, be then centrifugal Fe under the condition of 10000rpm 3O 4-HCPT@SiO 2Aqueous solution 30min, remove supernatant, adds washing with alcohol, ultrasonic dispersion 15-20min;
Seven, repeating step is six 3-4 time, and obtaining concentration is 7.5mg/mLFe 3O 4-HCPT@SiO 2Alcoholic solution;
Eight, by 10mLFe 3O 4-HCPT@SiO 2Alcoholic solution and 0.03ml aminopropyltriethoxywerene werene stirred 3 hours under the condition of 80 ℃, then according at rotating speed, being centrifugal 30min under the condition of 10000rpm, add ethanol, the order of ultrasonic dispersion 15-20min in precipitation, repeat 5 times, obtain the Fe that surface amination is modified 3O 4-HCPT@SiO 2Solution;
Nine, prepare golden nanometer particle:
To adding 0.01ml concentration in the 40mL distilled water, be that 0.25mol/L aqueous solution of chloraurate, 0.2mL concentration are that 0.2mol/L wet chemical, 0.6mL concentration are the 0.5mol/L sodium borohydride aqueous solution, under the condition of 600rpm, stir 10min, adding concentration is the sodium borohydride aqueous solution of 0.5mol/L again, until the bluish violet of solution becomes the orange red sodium borohydride aqueous solution that stops adding, stir again 5min, obtain solution of gold nanoparticles;
Ten, prepare strawberry shape gold nano compound particle:
Fe by the modification of 0.07ml surface amination 3O 4-HCPT@SiO 2Solution dilution is to 50ml, and then splashes in solution of gold nanoparticles, then, with the centrifugal 90min of the speed of 600rpm, obtains Au/SiO 2Solution;
11, prepare gold nanoshell:
The 25mg potassium carbonate is dissolved in the 100ml distilled water, and with the speed stirring 10min of 600rpm, then adding 1.5ml concentration is the aqueous solution of chloraurate of 0.25mol/L, continues to stir 30 minutes, obtains mixed liquor, by 4ml mixed liquor and Au/SiO 2Solution mixes, and then with the centrifugal 30min of 5000rpm, and then is scattered in 10mL water the precipitation after centrifugal ultrasonic 10min, obtains wrapping up the SiO of gold nanoshell 2The nanoparticle aqueous solution;
12, by 0.03g1,2-dimercapto-3-thiacyclohexane base Polyethylene Glycol and 3～4ml oxolane join in centrifuge tube, then add 1～5ml to wrap up in the SiO of gold nanoshell 2Nanoparticle aqueous solution and 50mg Porphyrin-Based Sensitizer molecule, reacted 72 hours, at rotating speed, is under the condition of 6000rpm centrifugal 10 minutes after reaction finishes, collecting precipitation, and precipitation is dispersed in methanol, obtain the Fe that concentration is 7.5mg/mL 3O 4-HCPT@SiO 2The methanol solution of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer;
13, by 50mg～100mg Fe 3O 4-HCPT@SiO 2Methanol solution, the 10mLN of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer, dinethylformamide and 50mg targeting biomolecule or antitumor drug were stirring at room reaction 2 hours, then at rotating speed, be that 14000rpm, temperature are centrifugal under the condition of 4 ℃, after collecting precipitation, be dispersed in methanol, obtain the methanol solution of targeting composite nanoparticle;
Porphyrin-Based Sensitizer molecule described in step 12 is 5-(4-chloroethene acyloxy phenyl)-10,15,20-tri-(4-carboxyl phenyl) porphyrin or tetrachloro acetoxyl group phenyl porphyrin;
Targeting biomolecule described in step 13 is folic acid;
Antitumor drug described in step 13 is monoclonal antibody Trastuzumab, paclitaxel, amycin, 5-fluorouracil or the cry of certain animals of first ammonia butterfly.
We are bright is adsorbed on Fe by polystyrolsulfon acid (PSS) and 10-hydroxycamptothecine self assembly mode 3O 4Magnetic nano-particle, at first the present invention has obtained a kind of efficient dewatering medicament drug-loading system; This method feasibility is high; Has Fe own 3O 4The physics targeting of magnetic nano-particle and the active targeting of further surface-functionalized modified biological molecule, the remarkable drug level that increases diseased region, under the condition that reaches the side effect minimum, have efficient treatment effect under the dual-target effect.This kind preparation method can solve the poor drug loading of medicine-carried system biocompatibility and targeting is low, the technical barrier of self assembly easily occurs medicine in transportation.
The other still a kind of novel targeting Integral rice corpuscles of this kind medicine-carried system: at Fe 3O 4-10-hydroxycamptothecine@SiO 2Coat gold nanoshell, first can pass through thermotherapy kill tumor cell and the tumor stem cell of gold nanoshell, not only can treat tumor and can suppress neoplasm metastasis; Second can improve the osmotic absorption of medicine by the permeability that the effect of thermotherapy chemical sensitization strengthens cell membrane, and the blood circulation that improves the tumor periphery increases medicine and enters inside tumor to improve the drug level ratio of tumor/normal structure.The Porphyrin-Based Sensitizer of the biomolecule folic acid connected by cystine linkage, make Fe 3O 4-10-hydroxycamptothecine@SiO 2@Au possesses the effect of photodynamics diagnosis and treatment.
The accompanying drawing explanation
Fig. 1 is the SEM image of the targeting composite nanoparticle of experiment one preparation;
Fig. 2 is that experiment one prepares step 2 gained Fe in the targeting composite nanoparticle 3O 4-HCPT nanoparticle TEM image;
Fig. 3 is the TEM image of the targeting composite nanoparticle of experiment one preparation;
Fig. 4 is the dynamic laser light scattering diagram picture of the targeting composite nanoparticle of monodispersed experiment one preparation in aqueous solution;
Fig. 5 is the spontaneous release accumulation of the medicine of targeting composite nanoparticle in the PBS solution of 37 ℃ of different pH value of experiment one preparation behavior curve, wherein
Figure GDA0000374297650000051
Represent that pH value is the spontaneous release accumulation of the medicine behavior curve in 9.2 the PBS solution of 37 ℃,
Figure GDA0000374297650000052
Represent that pH value is the spontaneous release accumulation of the medicine behavior curve in 8.0 the PBS solution of 37 ℃,
Figure GDA0000374297650000053
Represent that pH value is the spontaneous release accumulation of the medicine behavior curve in 7.4 the PBS solution of 37 ℃.
The specific embodiment
Technical solution of the present invention is not limited to the following cited specific embodiment, also comprises the combination in any between each specific embodiment.
The specific embodiment one: the preparation method of present embodiment targeting composite nanoparticle is carried out according to following steps:
One, prepare magnetic nano-particle:
By 10mL concentration, be the FeCl of 1mol/L 3Aqueous solution, 2.5mLFeSO 47H 2The HCl solution of O joins the NH that 125mL concentration is 0.7mol/L 37H 2O, continue to stir 30min, and centrifugalize is also collected the product in supernatant, by deionized water wash 3 times of the product in supernatant, and then is dispersed in the 150mL deionized water, obtains Fe 3O 4Nano-particle solution;
Described FeSO 47H 2FeSO in the HCl solution of O 47H 2The concentration of O is that the concentration of 2mol/L, HCl is 2mol/L;
Two, preparation Fe 3O 4-HCPT nanoparticle:
By 10mLFe 3O 4Nano-particle solution is distributed in the NaCl aqueous solution of the poly-hydrogen chloropropene amine of 10ml, ultrasonic reaction 30min, and magnetic separation, be then the NaCl solution washing of 0.51mol/L by concentration, finally is dispersed in water, obtaining concentration is the Fe of the PAH-parcel of 7.5mg/ml 3O 4Nano-particle solution;
In the NaCl aqueous solution of the poly-hydrogen chloropropene amine described in step 2, poly-hydrogen chloropropene amine concentration is 1mg/ml, and the concentration of NaCl is 0.5mol/L;
Three, by the Fe of 10mLPAH-parcel 3O 4Nano-particle solution is distributed to ultrasonic reaction 30min in the NaCl aqueous solution of 10ml polystyrolsulfon acid acid sodium, obtains Fe 3O 4The magnetic nano-particle precursor;
In the NaCl aqueous solution of the polystyrolsulfon acid acid sodium described in step 3, the concentration of kayexalate is 1mg/ml, and the concentration of NaCl is 0.5mol/L;
Four, the 364mg10-hydroxy camptothecin is dissolved in 150mLNaOH solution, regulating pH value is 11, then add concentration be the HCl of 1mol/L to regulate pH value be 4.8, obtain the HCPT microemulsion, by 10mlFe 3O 4The magnetic nano-particle precursor joins in the 10mLHCPT microemulsion, stir and pass through the uv absorption monitoring reaction, when the characteristic absorption peak of 10-hydroxycamptothecine disappears, stopped reaction, the magnetic separation collecting precipitation, and with pH value, be 4.8 HCl solution washing, then add in the 150mL deionized water, obtain the Fe of kayexalate/HCPT 3O 4The magnetic nano-particle aqueous solution;
Five, preparation Fe 3O 4-HCPT@SiO 2:
The 1mL ethyl orthosilicate is mixed with 30mL ethanol, obtain the alcoholic solution of ethyl orthosilicate, by 2.5mLNH 3H 2O, 38mLH 2The Fe of O, 38mL ethanol and 10mL kayexalate/HCPT 3O 4The magnetic nano-particle aqueous solution, then add the alcoholic solution of ethyl orthosilicate, reacted 4 hours, and magnetic separation, collecting precipitation, and the mixed solution of water and ethanol washing 3 times, redispersion, in the 100mL deionized water, obtains Fe 3O 4-HCPT@SiO 2Aqueous solution;
Six, at rotating speed, be then centrifugal Fe under the condition of 10000rpm 3O 4-HCPT@SiO 2Aqueous solution 30min, remove supernatant, adds washing with alcohol, ultrasonic dispersion 15-20min;
Seven, repeating step is six 3-4 time, and obtaining concentration is 7.5mg/mLFe 3O 4-HCPT@SiO 2Alcoholic solution;
Eight, by 10mLFe 3O 4-HCPT@SiO 2Alcoholic solution and 0.03ml aminopropyltriethoxywerene werene stirred 3 hours under the condition of 80 ℃, then according at rotating speed, being centrifugal 30min under the condition of 10000rpm, add ethanol, the order of ultrasonic dispersion 15-20min in precipitation, repeat 5 times, obtain the Fe that surface amination is modified 3O 4-HCPT@SiO 2Solution;
Nine, prepare golden nanometer particle:
To adding 0.01ml concentration in the 40mL distilled water, be that 0.25mol/L aqueous solution of chloraurate, 0.2mL concentration are that 0.2mol/L wet chemical, 0.6mL concentration are the 0.5mol/L sodium borohydride aqueous solution, under the condition of 600rpm, stir 10min, adding concentration is the sodium borohydride aqueous solution of 0.5mol/L again, until the bluish violet of solution becomes the orange red sodium borohydride aqueous solution that stops adding, stir again 5min, obtain solution of gold nanoparticles;
Ten, prepare strawberry shape gold nano compound particle:
Fe by the modification of 0.07ml surface amination 3O 4-HCPT@SiO 2Solution dilution is to 50ml, and then splashes in solution of gold nanoparticles, then, with the centrifugal 90min of the speed of 600rpm, obtains Au/SiO 2Solution;
11, prepare gold nanoshell:
The 25mg potassium carbonate is dissolved in the 100ml distilled water, and with the speed stirring 10min of 600rpm, then adding 1.5ml concentration is the aqueous solution of chloraurate of 0.25mol/L, continues to stir 30 minutes, obtains mixed liquor, by 4ml mixed liquor and Au/SiO 2Solution mixes, and then with the centrifugal 30min of 5000rpm, and then is scattered in 10mL water the precipitation after centrifugal ultrasonic 10min, obtains wrapping up the SiO of gold nanoshell 2The nanoparticle aqueous solution;
12, by 0.03g1,2-dimercapto-3-thiacyclohexane base Polyethylene Glycol and 3～4ml oxolane join in centrifuge tube, then add 1～5ml to wrap up in the SiO of gold nanoshell 2Nanoparticle aqueous solution and 50mg Porphyrin-Based Sensitizer molecule, reacted 72 hours, at rotating speed, is under the condition of 6000rpm centrifugal 10 minutes after reaction finishes, collecting precipitation, and precipitation is dispersed in methanol, obtain the Fe that concentration is 7.5mg/mL 3O 4-HCPT@SiO 2The methanol solution of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer;
13, by 50mg～100mg Fe 3O 4-HCPT@SiO 2Methanol solution, the 10mLN of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer, dinethylformamide and 50mg targeting biomolecule or antitumor drug were stirring at room reaction 2 hours, then at rotating speed, be that 14000rpm, temperature are centrifugal under the condition of 4 ℃, after collecting precipitation, be dispersed in methanol, obtain the methanol solution of targeting composite nanoparticle;
Porphyrin-Based Sensitizer molecule described in step 12 is 5-(4-chloroethene acyloxy phenyl)-10,15,20-tri-(4-carboxyl phenyl) porphyrin or tetrachloro acetoxyl group phenyl porphyrin;
Targeting biomolecule described in step 13 is folic acid;
Antitumor drug described in step 13 is monoclonal antibody Trastuzumab, paclitaxel, amycin, 5-fluorouracil or the cry of certain animals of first ammonia butterfly.
The specific embodiment two: present embodiment is different from the specific embodiment one be in step 12 by 0.03g1,2-dimercapto-3-thiacyclohexane base Polyethylene Glycol and 3.5ml oxolane join in centrifuge tube.Other is identical with the specific embodiment one.
The specific embodiment three: what present embodiment was different from the specific embodiment one is by 60mg～90mgFe in step 13 3O 4-HCPT@SiO 2Methanol solution, the 10mLN of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer, dinethylformamide and 50mg targeting biomolecule or antitumor drug were stirring at room reaction 2 hours.Other is identical with the specific embodiment one.
The specific embodiment four: what present embodiment was different from the specific embodiment one is by 70mg～80mgFe in step 13 3O 4-HCPT@SiO 2Methanol solution, the 10mLN of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer, dinethylformamide and 50mg targeting biomolecule or antitumor drug were stirring at room reaction 2 hours.Other is identical with the specific embodiment one.
The specific embodiment five: what present embodiment was different from the specific embodiment one is by 75mgFe in step 13 3O 4-HCPT@SiO 2Methanol solution, the 10mLN of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer, dinethylformamide and 50mg targeting biomolecule or antitumor drug were stirring at room reaction 2 hours.Other is identical with the specific embodiment one.
The specific embodiment six: the preparation method of present embodiment targeting composite nanoparticle is carried out according to following steps:
One, prepare magnetic nano-particle:
By 10mL concentration, be the FeCl of 1mol/L 3Aqueous solution, 2.5mLFeSO 47H 2The HCl solution of O joins the NH that 125mL concentration is 0.7mol/L 37H 2O, continue to stir 30min, and centrifugalize is also collected the product in supernatant, by deionized water wash 3 times of the product in supernatant, and then is dispersed in the 150mL deionized water, obtains Fe 3O 4Nano-particle solution;
Described FeSO 47H 2FeSO in the HCl solution of O 47H 2The concentration of O is that the concentration of 2mol/L, HCl is 2mol/L;
Two, preparation Fe 3O 4-HCPT nanoparticle:
By 10mlFe 3O 4Nano-particle solution is distributed in the NaCl aqueous solution of the poly-hydrogen chloropropene amine of 10ml, ultrasonic reaction 30min, and magnetic separation, be then the NaCl solution washing of 0.51mol/L by concentration, finally is dispersed in water, obtaining concentration is the Fe of the PAH-parcel of 7.5mg/ml 3O 4Nano-particle solution;
In the NaCl aqueous solution of the poly-hydrogen chloropropene amine described in step 2, poly-hydrogen chloropropene amine concentration is 1mg/ml, and the concentration of NaCl is 0.5mol/L;
Three, by the Fe of 10mLPAH-parcel 3O 4Nano-particle solution is distributed to ultrasonic reaction 30min in the NaCl aqueous solution of 10mL kayexalate, obtains Fe 3O 4The magnetic nano-particle precursor;
In the NaCl aqueous solution of the kayexalate described in step 3, the concentration of kayexalate is 1mg/ml, and the concentration of NaCl is 0.5mol/L;
Four, the 364mg10-hydroxy camptothecin is dissolved in 150mLNaOH solution, regulating pH value is 11, then add concentration be the HCl of 1mol/L to regulate pH value be 4.8, obtain the HCPT microemulsion, by 10mLFe 3O 4The magnetic nano-particle precursor joins in the 10mLHCPT microemulsion, stirs and passes through the uv absorption monitoring reaction, when the characteristic absorption peak of 10-hydroxycamptothecine disappears, stopped reaction, magnetic separation, and with pH value, be 4.8 HCl solution washing, the Fe of kayexalate/HCPT obtained 3O 4Magnetic nano-particle;
Five, by the Fe of kayexalate/HCPT 3O 4Magnetic nano-particle is distributed in the 150ml deionized water, obtains the Fe of kayexalate/HCPT 3O 4The magnetic nano-particle dispersion liquid, by the Fe of 10mL kayexalate/HCPT 3O 4The magnetic nano-particle dispersion liquid is distributed to ultrasonic reaction 30min in the NaCl aqueous solution of 10mL kayexalate, Fe 3O 4Magnetic nano-particle precursor a;
The 364mg10-hydroxy camptothecin is dissolved in 150mLNaOH solution, and regulating pH value is 11, then add concentration be the HCl of 1mol/L to regulate pH value be 4.8, obtain the HCPT microemulsion, by 10mLFe 3O 4Magnetic nano-particle precursor a joins in the 10mLHCPT microemulsion, stirs and passes through the uv absorption monitoring reaction, when the characteristic absorption peak of 10-hydroxycamptothecine disappears, stopped reaction, magnetic separation, and with pH value, be 4.8 HCl solution washing, the Fe of 2 strata Sodium styrene sulfonate/HCPT obtained 3O 4Magnetic nano-particle;
In the NaCl aqueous solution of described kayexalate, the concentration of kayexalate is 1mg/ml, and the concentration of NaCl is 0.5mol/L;
Six, repeating step five, then add in the 150mL deionized water, obtain the Fe of n+2 strata Sodium styrene sulfonate/HCPT 3O 4The magnetic nano-particle aqueous solution, wherein n is positive integer;
Seven, preparation Fe 3O 4-HCPT@SiO 2:
The 1mL ethyl orthosilicate is mixed with 30mL ethanol, obtain the alcoholic solution of ethyl orthosilicate, by 2.5mLNH 3H 2O, 38mLH 2The Fe of O, 38mL ethanol and 10mLn+2 strata Sodium styrene sulfonate/HCPT 3O 4The magnetic nano-particle aqueous solution, then add the alcoholic solution of ethyl orthosilicate, reacted 4 hours, and magnetic separation, collecting precipitation, and the mixed solution of water and ethanol washing 3 times, redispersion, in the 100mL deionized water, obtains Fe 3O 4-HCPT@SiO 2Aqueous solution;
Eight, at rotating speed, be then centrifugal Fe under the condition of 10000rpm 3O 4-HCPT@SiO 2Aqueous solution 30min, remove supernatant, adds appropriate washing with alcohol, ultrasonic dispersion 15-20min;
Nine, repeating step is eight 3-4 time, and obtaining concentration is 7.5mg/mLFe 3O 4-HCPT@SiO 2Alcoholic solution;
Ten, by 10mLFe 3O 4-HCPT@SiO 2Alcoholic solution and 0.03ml aminopropyltriethoxywerene werene stirred 3 hours under the condition of 80 ℃, then according at rotating speed, being centrifugal 30min under the condition of 10000rpm, add ethanol, the order of ultrasonic dispersion 15-20min in precipitation, repeat 5 times, obtain the Fe that surface amination is modified 3O 4-HCPT@SiO 2Solution;
11, prepare golden nanometer particle:
To adding 0.01ml concentration in the 40mL distilled water, be that 0.25mol/L aqueous solution of chloraurate, 0.2mL concentration are that 0.2mol/L wet chemical, 0.6mL concentration are the 0.5mol/L sodium borohydride aqueous solution, 600rpm stirs 10min, adding concentration is the sodium borohydride aqueous solution of 0.5mol/L again, until the bluish violet of solution becomes the orange red sodium borohydride aqueous solution that stops adding, stir again 5min, obtain solution of gold nanoparticles;
12, prepare strawberry shape gold nano compound particle:
Fe by the modification of 0.07ml surface amination 3O 4-HCPT@SiO 2Solution dilution is to 50ml, and then splashes in solution of gold nanoparticles, then, with the centrifugal 90min of the speed of 600rpm, obtains Au/SiO 2Solution;
13, prepare gold nanoshell:
The 25mg potassium carbonate is dissolved in the 100ml distilled water, and with the speed stirring 10min of 600rpm, then adding 1.5ml concentration is the aqueous solution of chloraurate of 0.25mol/L, continues to stir 30 minutes, obtains mixed liquor, by 4ml mixed liquor and Au/SiO 2Solution mixes, and then with the centrifugal 30min of 5000rpm, and then is scattered in 10mL water the precipitation after centrifugal ultrasonic 10min, obtains wrapping up the SiO of gold nanoshell 2The nanoparticle aqueous solution;
14, by 0.03g1,2-dimercapto-3-thiacyclohexane base Polyethylene Glycol and 3～4ml oxolane join in centrifuge tube, then add 1～5ml to wrap up in the SiO of gold nanoshell 2Nanoparticle aqueous solution and 50mg Porphyrin-Based Sensitizer molecule, reacted 72 hours, at rotating speed, is under the condition of 6000rpm centrifugal 10 minutes after reaction finishes, collecting precipitation, and precipitation is dispersed in methanol, obtain the Fe that concentration is 7.5mg/mL 3O 4-HCPT@SiO 2The Integral rice corpuscles of coupling Porphyrin-Based Sensitizer;
15, by 50mg～100mg Fe 3O 4-HCPT@SiO 2Methanol solution, the 10mLN of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer, dinethylformamide and 50mg targeting biomolecule or antitumor drug were stirring at room reaction 2 hours, then at rotating speed, be that 14000rpm, temperature are centrifugal under the condition of 4 ℃, after collecting precipitation, be dispersed in methanol, obtain the methanol solution of targeting composite nanoparticle;
Porphyrin-Based Sensitizer molecule described in step 14 is 5-(4-chloroethene acyloxy phenyl)-10,15,20-tri-(4-carboxyl phenyl) porphyrin or tetrachloro acetoxyl group phenyl porphyrin;
Targeting biomolecule described in step 15 is folic acid;
Antitumor drug described in step 15 is monoclonal antibody Trastuzumab, paclitaxel, amycin, 5-fluorouracil or the cry of certain animals of first ammonia butterfly.
The specific embodiment seven: present embodiment is different from the specific embodiment six be in step 14 by 0.03g1,2-dimercapto-3-thiacyclohexane base Polyethylene Glycol and 3.5ml oxolane join in centrifuge tube.Other is identical with the specific embodiment six.
The specific embodiment eight: what present embodiment was different from the specific embodiment six is by 60mg～90mgFe in step 15 3O 4-HCPT@SiO 2Methanol solution, the 10mLN of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer, dinethylformamide and 50mg targeting biomolecule or antitumor drug were stirring at room reaction 2 hours.Other is identical with the specific embodiment six.
The specific embodiment nine: what present embodiment was different from the specific embodiment six is by 70mg～80mgFe in step 15 3O 4-HCPT@SiO 2Methanol solution, the 10mLN of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer, dinethylformamide and 50mg targeting biomolecule or antitumor drug were stirring at room reaction 2 hours.Other is identical with the specific embodiment six.
The specific embodiment ten: what present embodiment was different from the specific embodiment six is by 75mgFe in step 15 3O 4-HCPT@SiO 2Methanol solution, the 10mLN of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer, dinethylformamide and 50mg targeting biomolecule or antitumor drug were stirring at room reaction 2 hours.Other is identical with the specific embodiment six.
Adopt following experimental verification effect of the present invention:
Experiment one:
The preparation method of targeting composite nanoparticle is carried out according to following steps:
One, prepare magnetic nano-particle:
By 10mL concentration, be the FeCl of 1mol/L 3Aqueous solution, 2.5mLFeSO 47H 2The HCl solution of O joins the NH that 125mL concentration is 0.7mol/L 37H 2O, continue to stir 30min, and centrifugalize is also collected the product in supernatant, by deionized water wash 3 times of the product in supernatant, and then is dispersed in the 150mL deionized water, obtains Fe 3O 4Nano-particle solution;
Described FeSO 47H 2FeSO in the HCl solution of O 47H 2The concentration of O is that the concentration of 2mol/L, HCl is 2mol/L;
Two, preparation Fe 3O 4-HCPT nanoparticle:
By 10mLFe 3O 4Nano-particle solution is distributed in the NaCl aqueous solution of the poly-hydrogen chloropropene amine of 10ml, ultrasonic reaction 30min, and magnetic separation, be then the NaCl solution washing of 0.51mol/L by concentration, finally is dispersed in water, obtaining concentration is the Fe of the PAH-parcel of 7.5mg/ml 3O 4Nano-particle solution;
In the NaCl aqueous solution of the poly-hydrogen chloropropene amine described in step 2, poly-hydrogen chloropropene amine concentration is 1mg/ml, and the concentration of NaCl is 0.5mol/L;
Three, by the Fe of 10mLPAH-parcel 3O 4Nano-particle solution is distributed to ultrasonic reaction 30min in the NaCl aqueous solution of 10ml polystyrolsulfon acid acid sodium, obtains Fe 3O 4The magnetic nano-particle precursor;
In the NaCl aqueous solution of the polystyrolsulfon acid acid sodium described in step 3, the concentration of kayexalate is 1mg/ml, and the concentration of NaCl is 0.5mol/L;
Four, the 364mg10-hydroxy camptothecin is dissolved in 150mLNaOH solution, regulating pH value is 11, then add concentration be the HCl of 1mol/L to regulate pH value be 4.8, obtain the HCPT microemulsion, by 10mlFe 3O 4The magnetic nano-particle precursor joins in the 10mLHCPT microemulsion, stir and pass through the uv absorption monitoring reaction, when the characteristic absorption peak of 10-hydroxycamptothecine disappears, stopped reaction, the magnetic separation collecting precipitation, and with pH value, be 4.8 HCl solution washing, then add in the 150mL deionized water, obtain the Fe of kayexalate/HCPT 3O 4The magnetic nano-particle aqueous solution;
Five, preparation Fe 3O 4-HCPT@SiO 2:
The 1mL ethyl orthosilicate is mixed with 30mL ethanol, obtain the alcoholic solution of ethyl orthosilicate, by 2.5mLNH 3H 2O, 38mLH 2The Fe of O, 38mL ethanol and 10mL kayexalate/HCPT 3O 4The magnetic nano-particle aqueous solution, then add the alcoholic solution of ethyl orthosilicate, reacted 4 hours, and magnetic separation, collecting precipitation, and the mixed solution of water and ethanol washing 3 times, redispersion, in the 100mL deionized water, obtains Fe 3O 4-HCPT@SiO 2Aqueous solution;
Six, at rotating speed, be then centrifugal Fe under the condition of 10000rpm 3O 4-HCPT@SiO 2Aqueous solution 30min, remove supernatant, adds washing with alcohol, ultrasonic dispersion 20min;
Seven, repeating step is 63 times, and obtaining concentration is 7.5mg/mLFe 3O 4-HCPT@SiO 2Alcoholic solution;
Eight, by 10mLFe 3O 4-HCPT@SiO 2Alcoholic solution and 0.03ml aminopropyltriethoxywerene werene stirred 3 hours under the condition of 80 ℃, then according at rotating speed, being centrifugal 30min under the condition of 10000rpm, add ethanol, the order of ultrasonic dispersion 15-20min in precipitation, repeat 5 times, obtain the Fe that surface amination is modified 3O 4-HCPT@SiO 2Solution;
Nine, prepare golden nanometer particle:
To adding 0.01ml concentration in the 40mL distilled water, be that 0.25mol/L aqueous solution of chloraurate, 0.2mL concentration are that 0.2mol/L wet chemical, 0.6mL concentration are the 0.5mol/L sodium borohydride aqueous solution, under the condition of 600rpm, stir 10min, adding concentration is the sodium borohydride aqueous solution of 0.5mol/L again, until the bluish violet of solution becomes the orange red sodium borohydride aqueous solution that stops adding, stir again 5min, obtain solution of gold nanoparticles;
Ten, prepare strawberry shape gold nano compound particle:
Fe by the modification of 0.07ml surface amination 3O 4-HCPT@SiO 2Solution dilution is to 50ml, and then splashes in solution of gold nanoparticles, then, with the centrifugal 90min of the speed of 600rpm, obtains Au/SiO 2Solution;
11, prepare gold nanoshell:
The 25mg potassium carbonate is dissolved in the 100ml distilled water, and with the speed stirring 10min of 600rpm, then adding 1.5ml concentration is the aqueous solution of chloraurate of 0.25mol/L, continues to stir 30 minutes, obtains mixed liquor, by 4ml mixed liquor and Au/SiO 2Solution mixes, and then with the centrifugal 30min of 5000rpm, and then is scattered in 10mL water the precipitation after centrifugal ultrasonic 10min, obtains wrapping up the SiO of gold nanoshell 2The nanoparticle aqueous solution;
12, by 0.03g1,2-dimercapto-3-thiacyclohexane base Polyethylene Glycol and 4ml oxolane join in centrifuge tube, then add 5ml to wrap up in the SiO of gold nanoshell 2Nanoparticle aqueous solution and 50mg Porphyrin-Based Sensitizer molecule, reacted 72 hours, at rotating speed, is under the condition of 6000rpm centrifugal 10 minutes after reaction finishes, collecting precipitation, and precipitation is dispersed in methanol, obtain the Fe that concentration is 7.5mg/mL 3O 4-HCPT@SiO 2The methanol solution of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer;
13, by 50mg Fe 3O 4-HCPT@SiO 2Methanol solution, the 10mLN of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer, dinethylformamide and 50mg targeting biomolecule were stirring at room reaction 2 hours, then at rotating speed, be that 14000rpm, temperature are centrifugal under the condition of 4 ℃, after collecting precipitation, be dispersed in methanol, obtain the methanol solution of targeting composite nanoparticle;
Porphyrin-Based Sensitizer molecule described in step 12 is 5-(4-chloroethene acyloxy phenyl)-10,15,20-tri-(4-carboxyl phenyl) porphyrin;
Targeting biomolecule described in step 13 is folic acid.
Experiment two:
The preparation method of targeting composite nanoparticle is carried out according to following steps:
One, prepare magnetic nano-particle:
By 10mL concentration, be the FeCl of 1mol/L 3Aqueous solution, 2.5mLFeSO 47H 2The HCl solution of O joins the NH that 125mL concentration is 0.7mol/L 37H 2O, continue to stir 30min, and centrifugalize is also collected the product in supernatant, by deionized water wash 3 times of the product in supernatant, and then is dispersed in the 150mL deionized water, obtains Fe 3O 4Nano-particle solution;
Described FeSO 47H 2FeSO in the HCl solution of O 47H 2The concentration of O is that the concentration of 2mol/L, HCl is 2mol/L;
Two, preparation Fe 3O 4-HCPT nanoparticle:
By 10mlFe 3O 4Nano-particle solution is distributed in the NaCl aqueous solution of the poly-hydrogen chloropropene amine of 10ml, ultrasonic reaction 30min, and magnetic separation, be then the NaCl solution washing of 0.51mol/L by concentration, finally is dispersed in water, obtaining concentration is the Fe of the PAH-parcel of 7.5mg/ml 3O 4Nano-particle solution;
In the NaCl aqueous solution of the poly-hydrogen chloropropene amine described in step 2, poly-hydrogen chloropropene amine concentration is 1mg/ml, and the concentration of NaCl is 0.5mol/L;
Three, by the Fe of 10mLPAH-parcel 3O 4Nano-particle solution is distributed to ultrasonic reaction 30min in the NaCl aqueous solution of 10mL kayexalate, obtains Fe 3O 4The magnetic nano-particle precursor;
In the NaCl aqueous solution of the kayexalate described in step 3, the concentration of kayexalate is 1mg/ml, and the concentration of NaCl is 0.5mol/L;
Four, the 364mg10-hydroxy camptothecin is dissolved in 150mLNaOH solution, regulating pH value is 11, then add concentration be the HCl of 1mol/L to regulate pH value be 4.8, obtain the HCPT microemulsion, by 10mLFe 3O 4The magnetic nano-particle precursor joins in the 10mLHCPT microemulsion, stirs and passes through the uv absorption monitoring reaction, when the characteristic absorption peak of 10-hydroxycamptothecine disappears, stopped reaction, magnetic separation, and with pH value, be 4.8 HCl solution washing, the Fe of kayexalate/HCPT obtained 3O 4Magnetic nano-particle;
Five, by the Fe of kayexalate/HCPT 3O 4Magnetic nano-particle is distributed in the 150ml deionized water, obtains the Fe of kayexalate/HCPT 3O 4The magnetic nano-particle dispersion liquid, by the Fe of 10mL kayexalate/HCPT 3O 4The magnetic nano-particle dispersion liquid is distributed to ultrasonic reaction 30min in the NaCl aqueous solution of 10mL kayexalate, Fe 3O 4Magnetic nano-particle precursor a;
The 364mg10-hydroxy camptothecin is dissolved in 150mLNaOH solution, and regulating pH value is 11, then add concentration be the HCl of 1mol/L to regulate pH value be 4.8, obtain the HCPT microemulsion, by 10mLFe 3O 4Magnetic nano-particle precursor a joins in the 10mLHCPT microemulsion, stirs and passes through the uv absorption monitoring reaction, when the characteristic absorption peak of 10-hydroxycamptothecine disappears, stopped reaction, magnetic separation, and with pH value, be 4.8 HCl solution washing, the Fe of 2 strata Sodium styrene sulfonate/HCPT obtained 3O 4Magnetic nano-particle;
In the NaCl aqueous solution of described kayexalate, the concentration of kayexalate is 1mg/ml, and the concentration of NaCl is 0.5mol/L;
Six, repeating step five, then add in the 150mL deionized water, obtain the Fe of n+2 strata Sodium styrene sulfonate/HCPT 3O 4The magnetic nano-particle aqueous solution, wherein n is positive integer;
Seven, preparation Fe 3O 4-HCPT@SiO 2:
The 1mL ethyl orthosilicate is mixed with 30mL ethanol, obtain the alcoholic solution of ethyl orthosilicate, by 2.5mLNH 3H 2O, 38mLH 2The Fe of O, 38mL ethanol and 10mLn+2 strata Sodium styrene sulfonate/HCPT 3O 4The magnetic nano-particle aqueous solution, then add the alcoholic solution of ethyl orthosilicate, reacted 4 hours, and magnetic separation, collecting precipitation, and the mixed solution of water and ethanol washing 3 times, redispersion, in the 100mL deionized water, obtains Fe 3O 4-HCPT@SiO 2Aqueous solution;
Eight, at rotating speed, be then centrifugal Fe under the condition of 10000rpm 3O 4-HCPT@SiO 2Aqueous solution 30min, remove supernatant, adds appropriate washing with alcohol, ultrasonic dispersion 15-20min;
Nine, repeating step is 84 times, and obtaining concentration is 7.5mg/mLFe 3O 4-HCPT@SiO 2Alcoholic solution;
Ten, by 10mLFe 3O 4-HCPT@SiO 2Alcoholic solution and 0.03ml aminopropyltriethoxywerene werene stirred 3 hours under the condition of 80 ℃, then according at rotating speed, being centrifugal 30min under the condition of 10000rpm, add ethanol, the order of ultrasonic dispersion 15-20min in precipitation, repeat 5 times, obtain the Fe that surface amination is modified 3O 4-HCPT@SiO 2Solution;
11, prepare golden nanometer particle:
To adding 0.01ml concentration in the 40mL distilled water, be that 0.25mol/L aqueous solution of chloraurate, 0.2mL concentration are that 0.2mol/L wet chemical, 0.6mL concentration are the 0.5mol/L sodium borohydride aqueous solution, 600rpm stirs 10min, adding concentration is the sodium borohydride aqueous solution of 0.5mol/L again, until the bluish violet of solution becomes the orange red sodium borohydride aqueous solution that stops adding, stir again 5min, obtain solution of gold nanoparticles;
12, prepare strawberry shape gold nano compound particle:
Fe by the modification of 0.07ml surface amination 3O 4-HCPT@SiO 2Solution dilution is to 50ml, and then splashes in solution of gold nanoparticles, then, with the centrifugal 90min of the speed of 600rpm, obtains Au/SiO 2Solution;
13, prepare gold nanoshell:
The 25mg potassium carbonate is dissolved in the 100ml distilled water, and with the speed stirring 10min of 600rpm, then adding 1.5ml concentration is the aqueous solution of chloraurate of 0.25mol/L, continues to stir 30 minutes, obtains mixed liquor, by 4ml mixed liquor and Au/SiO 2Solution mixes, and then with the centrifugal 30min of 5000rpm, and then is scattered in 10mL water the precipitation after centrifugal ultrasonic 10min, obtains wrapping up the SiO of gold nanoshell 2The nanoparticle aqueous solution;
14, by 0.03g1,2-dimercapto-3-thiacyclohexane base Polyethylene Glycol and 3ml oxolane join in centrifuge tube, then add 4ml to wrap up in the SiO of gold nanoshell 2Nanoparticle aqueous solution and 50mg Porphyrin-Based Sensitizer molecule, reacted 72 hours, at rotating speed, is under the condition of 6000rpm centrifugal 10 minutes after reaction finishes, collecting precipitation, and precipitation is dispersed in methanol, obtain the Fe that concentration is 7.5mg/mL 3O 4-HCPT@SiO 2The Integral rice corpuscles of coupling Porphyrin-Based Sensitizer;
15, by 80mgFe 3O 4-HCPT@SiO 2Methanol solution, the 10mLN of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer, dinethylformamide and 50mg antitumor drug were stirring at room reaction 2 hours, then at rotating speed, be that 14000rpm, temperature are centrifugal under the condition of 4 ℃, after collecting precipitation, be dispersed in methanol, obtain the methanol solution of targeting composite nanoparticle;
Porphyrin-Based Sensitizer molecule described in step 14 is tetrachloro acetoxyl group phenyl porphyrin;
Antitumor drug described in step 15 is the monoclonal antibody Trastuzumab.

Claims (10)

1. the preparation method of targeting composite nanoparticle is characterized in that the preparation method of targeting composite nanoparticle is carried out according to following steps:
One, prepare magnetic nano-particle:
By 10ml concentration, be the FeCl of 1mol/L 3Aqueous solution, 2.5ml FeSO 47H 2The HCl solution of O joins the NH that 125ml concentration is 0.7mol/L 37H 2O, continue to stir 30min, and centrifugalize is also collected the product in supernatant, by deionized water wash 3 times of the product in supernatant, and then is dispersed in the 150ml deionized water, obtains Fe 3O 4Nano-particle solution;
Described FeSO 47H 2FeSO in the HCl solution of O 47H 2The concentration of O is that the concentration of 2mol/L, HCl is 2mol/L;
Two, preparation Fe 3O 4-HCPT nanoparticle:
By 10ml Fe 3O 4Nano-particle solution is distributed in the NaCl aqueous solution of the poly-hydrogen chloropropene amine PAH of 10ml, ultrasonic reaction 30min, and magnetic separation, be then the NaCl solution washing of 0.51mol/L by concentration, finally is dispersed in water, obtaining concentration is the Fe of the PAH-parcel of 7.5mg/ml 3O 4Nano-particle solution;
In the NaCl aqueous solution of the poly-hydrogen chloropropene amine described in step 2, poly-hydrogen chloropropene amine concentration is 1mg/ml, and the concentration of NaCl is 0.5mol/L;
Three, by the Fe of 10ml PAH-parcel 3O 4Nano-particle solution is distributed to ultrasonic reaction 30min in the NaCl aqueous solution of 10ml kayexalate, obtains Fe 3O 4The magnetic nano-particle precursor;
In the NaCl aqueous solution of the polystyrolsulfon acid acid sodium described in step 3, the concentration of kayexalate is 1mg/ml, and the concentration of NaCl is 0.5mol/L;
Four, 364mg10-hydroxy camptothecin HCPT is dissolved in 150ml NaOH solution, regulating pH value is 11, then add concentration be the HCl of 1mol/L to regulate pH value be 4.8, obtain the HCPT microemulsion, by 10mlFe 3O 4The magnetic nano-particle precursor joins in 10ml HCPT microemulsion, stir and pass through the uv absorption monitoring reaction, when the characteristic absorption peak of 10-hydroxycamptothecine disappears, stopped reaction, the magnetic separation collecting precipitation, and with pH value, be 4.8 HCl solution washing, then add in the 150mL deionized water, obtain the Fe of kayexalate/HCPT 3O 4The magnetic nano-particle aqueous solution;
Five, preparation Fe 3O 4-HCPT@SiO 2:
The 1ml ethyl orthosilicate is mixed with 30ml ethanol, obtain the alcoholic solution of ethyl orthosilicate, by 2.5mLNH 3H 2O, 38mL H 2The Fe of O, 38mL ethanol and 10mL kayexalate/HCPT 3O 4The magnetic nano-particle aqueous solution, then add the alcoholic solution of ethyl orthosilicate, reacted 4 hours, and magnetic separation, collecting precipitation, and the mixed solution of water and ethanol washing 3 times, redispersion, in the 100ml deionized water, obtains Fe 3O 4-HCPT@SiO 2Aqueous solution;
Six, at rotating speed, be then centrifugal Fe under the condition of 10000rpm 3O 4-HCPT@SiO 2Aqueous solution 30min, remove supernatant, adds washing with alcohol, ultrasonic dispersion 15-20min;
Seven, repeating step is six 3-4 time, and obtaining concentration is 7.5mg/mLFe 3O 4-HCPT@SiO 2Alcoholic solution;
Eight, by 10ml Fe 3O 4-HCPT@SiO 2Alcoholic solution and 0.03ml aminopropyltriethoxywerene werene stirred 3 hours under the condition of 80 ℃, then according at rotating speed, being centrifugal 30min under the condition of 10000rpm, add ethanol, the order of ultrasonic dispersion 15-20min in precipitation, repeat 5 times, obtain the Fe that surface amination is modified 3O 4-HCPT@SiO 2Solution;
Nine, prepare golden nanometer particle:
To adding 0.01ml concentration in the 40ml distilled water, be that 0.25mol/L aqueous solution of chloraurate, 0.2ml concentration are that 0.2mol/L wet chemical, 0.6ml concentration are the 0.5mol/L sodium borohydride aqueous solution, under the condition of 600rpm, stir 10min, adding concentration is the sodium borohydride aqueous solution of 0.5mol/L again, until the bluish violet of solution becomes the orange red sodium borohydride aqueous solution that stops adding, stir again 5min, obtain solution of gold nanoparticles;
Ten, prepare strawberry shape gold nano compound particle:
Fe by the modification of 0.07ml surface amination 3O 4-HCPT@SiO 2Solution dilution is to 50ml, and then splashes in solution of gold nanoparticles, then, with the centrifugal 90min of the speed of 600rpm, obtains Au/SiO 2Solution;
11, prepare gold nanoshell:
The 25mg potassium carbonate is dissolved in the 100ml distilled water, and with the speed stirring 10min of 600rpm, then adding 1.5ml concentration is the aqueous solution of chloraurate of 0.25mol/L, continues to stir 30 minutes, obtains mixed liquor, by 4ml mixed liquor and Au/SiO 2Solution mixes, and then with the centrifugal 30min of 5000rpm, and then is scattered in 10ml water the precipitation after centrifugal ultrasonic 10min, obtains wrapping up the SiO of gold nanoshell 2The nanoparticle aqueous solution;
12, by 0.03g1,2-dimercapto-3-thiacyclohexane base Polyethylene Glycol and 3～4ml oxolane join in centrifuge tube, then add 1～5ml to wrap up in the SiO of gold nanoshell 2Nanoparticle aqueous solution and 50mg Porphyrin-Based Sensitizer molecule, reacted 72 hours, at rotating speed, is under the condition of 6000rpm centrifugal 10 minutes after reaction finishes, collecting precipitation, and precipitation is dispersed in methanol, obtain the Fe that concentration is 7.5mg/mL 3O 4-HCPT@SiO 2The methanol solution of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer;
13, by 50mg～100mg Fe 3O 4-HCPT@SiO 2The methanol solution of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer, 10ml N, dinethylformamide and 50mg targeting biomolecule or antitumor drug were stirring at room reaction 2 hours, then at rotating speed, be that 14000rpm, temperature are centrifugal under the condition of 4 ℃, after collecting precipitation, be dispersed in methanol, obtain the methanol solution of targeting composite nanoparticle;
Porphyrin-Based Sensitizer molecule described in step 12 is 5-(4-chloroethene acyloxy phenyl)-10,15,20-tri-(4-carboxyl phenyl) porphyrin or tetrachloro acetoxyl group phenyl porphyrin;
Targeting biomolecule described in step 13 is folic acid;
Antitumor drug described in step 13 is monoclonal antibody Trastuzumab, paclitaxel, amycin, 5-fluorouracil or methotrexate.
2. the preparation method of targeting composite nanoparticle according to claim 1, is characterized in that in step 12 that by 0.03g1,2-dimercapto-3-thiacyclohexane base Polyethylene Glycol and 3.5ml oxolane join in centrifuge tube.
3. the preparation method of targeting composite nanoparticle according to claim 1, is characterized in that in step 13 60mg～90mg Fe 3O 4-HCPT@SiO 2Methanol solution, the 10mlN of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer, dinethylformamide and 50mg targeting biomolecule or antitumor drug were stirring at room reaction 2 hours.
4. the preparation method of targeting composite nanoparticle according to claim 1, is characterized in that in step 13 70mg～80mg Fe 3O 4-HCPT@SiO 2Methanol solution, the 10mlN of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer, dinethylformamide and 50mg targeting biomolecule or antitumor drug were stirring at room reaction 2 hours.
5. the preparation method of targeting composite nanoparticle according to claim 1, is characterized in that in step 13 75mgFe 3O 4-HCPT@SiO 2The methanol solution of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer, 10ml DMF and 50mg targeting biomolecule or antitumor drug were stirring at room reaction 2 hours.
6. the preparation method of targeting composite nanoparticle is characterized in that the preparation method of targeting composite nanoparticle is carried out according to following steps:
One, prepare magnetic nano-particle:
By 10ml concentration, be the FeCl of 1mol/L 3Aqueous solution, 2.5ml FeSO 47H 2The HCl solution of O joins the NH that 125ml concentration is 0.7mol/L 37H 2O, continue to stir 30min, and centrifugalize is also collected the product in supernatant, by deionized water wash 3 times of the product in supernatant, and then is dispersed in the 150ml deionized water, obtains Fe 3O 4Nano-particle solution;
Described FeSO 47H 2FeSO in the HCl solution of O 47H 2The concentration of O is that the concentration of 2mol/L, HCl is 2mol/L;
Two, preparation Fe 3O 4-HCPT nanoparticle:
By 10mlFe 3O 4Nano-particle solution is distributed in the NaCl aqueous solution of the poly-hydrogen chloropropene amine of 10ml, ultrasonic reaction 30min, and magnetic separation, be then the NaCl solution washing of 0.51mol/L by concentration, finally is dispersed in water, obtaining concentration is the Fe of the PAH-parcel of 7.5mg/ml 3O 4Nano-particle solution;
In the NaCl aqueous solution of the poly-hydrogen chloropropene amine described in step 2, poly-hydrogen chloropropene amine concentration is 1mg/ml, and the concentration of NaCl is 0.5mol/L;
Three, by the Fe of 10ml PAH-parcel 3O 4Nano-particle solution is distributed to ultrasonic reaction 30min in the NaCl aqueous solution of 10ml kayexalate, obtains Fe 3O 4The magnetic nano-particle precursor;
In the NaCl aqueous solution of the kayexalate described in step 3, the concentration of kayexalate is 1mg/ml, and the concentration of NaCl is 0.5mol/L;
Four, the 364mg10-hydroxy camptothecin is dissolved in 150ml NaOH solution, regulating pH value is 11, then add concentration be the HCl of 1mol/L to regulate pH value be 4.8, obtain the HCPT microemulsion, by 10ml Fe 3O 4The magnetic nano-particle precursor joins in 10ml HCPT microemulsion, stirs and passes through the uv absorption monitoring reaction, when the characteristic absorption peak of 10-hydroxycamptothecine disappears, stopped reaction, magnetic separation, and with pH value, be 4.8 HCl solution washing, the Fe of kayexalate/HCPT obtained 3O 4Magnetic nano-particle;
Five, by the Fe of kayexalate/HCPT 3O 4Magnetic nano-particle is distributed in the 150ml deionized water, obtains the Fe of kayexalate/HCPT 3O 4The magnetic nano-particle dispersion liquid, by the Fe of 10ml kayexalate/HCPT 3O 4The magnetic nano-particle dispersion liquid is distributed to ultrasonic reaction 30min in the NaCl aqueous solution of 10ml kayexalate, Fe 3O 4Magnetic nano-particle precursor a;
The 364mg10-hydroxy camptothecin is dissolved in 150ml NaOH solution, and regulating pH value is 11, then add concentration be the HCl of 1mol/L to regulate pH value be 4.8, obtain the HCPT microemulsion, by 10ml Fe 3O 4Magnetic nano-particle precursor a joins in 10ml HCPT microemulsion, stirs and passes through the uv absorption monitoring reaction, when the characteristic absorption peak of 10-hydroxycamptothecine disappears, stopped reaction, magnetic separation, and with pH value, be 4.8 HCl solution washing, the Fe of 2 strata Sodium styrene sulfonate/HCPT obtained 3O 4Magnetic nano-particle;
In the NaCl aqueous solution of described kayexalate, the concentration of kayexalate is 1mg/ml, and the concentration of NaCl is 0.5mol/L;
Six, repeating step five, then add in the 150mL deionized water, obtain the Fe of n+2 strata Sodium styrene sulfonate/HCPT 3O 4The magnetic nano-particle aqueous solution, wherein n is positive integer;
Seven, preparation Fe 3O 4-HCPT@SiO 2:
The 1ml ethyl orthosilicate is mixed with 30ml ethanol, obtain the alcoholic solution of ethyl orthosilicate, by 2.5mLNH 3H 2O, 38mL H 2The Fe of O, 38mL ethanol and 10mL n+2 strata Sodium styrene sulfonate/HCPT 3O 4The magnetic nano-particle aqueous solution, then add the alcoholic solution of ethyl orthosilicate, reacted 4 hours, and magnetic separation, collecting precipitation, and the mixed solution of water and ethanol washing 3 times, redispersion, in the 100ml deionized water, obtains Fe 3O 4-HCPT@SiO 2Aqueous solution;
Eight, at rotating speed, be then centrifugal Fe under the condition of 10000rpm 3O 4-HCPT@SiO 2Aqueous solution 30min, remove supernatant, adds appropriate washing with alcohol, ultrasonic dispersion 15-20min;
Nine, repeating step is eight 3-4 time, and obtaining concentration is 7.5mg/mL Fe 3O 4-HCPT@SiO 2Alcoholic solution;
Ten, by 10ml Fe 3O 4-HCPT@SiO 2Alcoholic solution and 0.03ml aminopropyltriethoxywerene werene stirred 3 hours under the condition of 80 ℃, then according at rotating speed, being centrifugal 30min under the condition of 10000rpm, add ethanol, the order of ultrasonic dispersion 15-20min in precipitation, repeat 5 times, obtain the Fe that surface amination is modified 3O 4-HCPT@SiO 2Solution;
11, prepare golden nanometer particle:
To adding 0.01ml concentration in the 40ml distilled water, be that 0.25mol/L aqueous solution of chloraurate, 0.2ml concentration are that 0.2mol/L wet chemical, 0.6ml concentration are the 0.5mol/L sodium borohydride aqueous solution, 600rpm stirs 10min, adding concentration is the sodium borohydride aqueous solution of 0.5mol/L again, until the bluish violet of solution becomes the orange red sodium borohydride aqueous solution that stops adding, stir again 5min, obtain solution of gold nanoparticles;
12, prepare strawberry shape gold nano compound particle:
Fe by the modification of 0.07ml surface amination 3O 4-HCPT@SiO 2Solution dilution is to 50ml, and then splashes in solution of gold nanoparticles, then, with the centrifugal 90min of the speed of 600rpm, obtains Au/SiO 2Solution;
13, prepare gold nanoshell:
The 25mg potassium carbonate is dissolved in the 100ml distilled water, and with the speed stirring 10min of 600rpm, then adding 1.5ml concentration is the aqueous solution of chloraurate of 0.25mol/L, continues to stir 30 minutes, obtains mixed liquor, by 4ml mixed liquor and Au/SiO 2Solution mixes, and then with the centrifugal 30min of 5000rpm, and then is scattered in 10ml water the precipitation after centrifugal ultrasonic 10min, obtains wrapping up the SiO of gold nanoshell 2The nanoparticle aqueous solution;
14, by 0.03g1,2-dimercapto-3-thiacyclohexane base Polyethylene Glycol and 3～4ml oxolane join in centrifuge tube, then add 1～5ml to wrap up in the SiO of gold nanoshell 2Nanoparticle aqueous solution and 50mg Porphyrin-Based Sensitizer molecule, reacted 72 hours, at rotating speed, is under the condition of 6000rpm centrifugal 10 minutes after reaction finishes, collecting precipitation, and precipitation is dispersed in methanol, obtain the Fe that concentration is 7.5mg/mL 3O 4-HCPT@SiO 2The methanol solution of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer;
15, by 50mg～100mg Fe 3O 4-HCPT@SiO 2The methanol solution of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer, 10ml N, dinethylformamide and 50mg targeting biomolecule or antitumor drug were stirring at room reaction 2 hours, then at rotating speed, be that 14000rpm, temperature are centrifugal under the condition of 4 ℃, after collecting precipitation, be dispersed in methanol, obtain the methanol solution of targeting composite nanoparticle;
Porphyrin-Based Sensitizer molecule described in step 14 is 5-(4-chloroethene acyloxy phenyl)-10,15,20-tri-(4-carboxyl phenyl) porphyrin or tetrachloro acetoxyl group phenyl porphyrin;
Targeting biomolecule described in step 15 is folic acid;
Antitumor drug described in step 15 is monoclonal antibody Trastuzumab, paclitaxel, amycin, 5-fluorouracil or the cry of certain animals of first ammonia butterfly.
7. the preparation method of targeting composite nanoparticle according to claim 6, is characterized in that in step 14 that by 0.03g1,2-dimercapto-3-thiacyclohexane base Polyethylene Glycol and 3.5ml oxolane join in centrifuge tube.
8. the preparation method of targeting composite nanoparticle according to claim 6, is characterized in that in step 15 60mg～90mg Fe 3O 4-HCPT@SiO 2Methanol solution, the 10mlN of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer, dinethylformamide and 50mg targeting biomolecule or antitumor drug were stirring at room reaction 2 hours.
9. the preparation method of targeting composite nanoparticle according to claim 6, is characterized in that in step 15 70mg～80mg Fe 3O 4-HCPT@SiO 2Methanol solution, the 10mlN of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer, dinethylformamide and 50mg targeting biomolecule or antitumor drug were stirring at room reaction 2 hours.
10. the preparation method of targeting composite nanoparticle according to claim 6, is characterized in that in step 15 75mgFe 3O 4-HCPT@SiO 2The methanol solution of the Integral rice corpuscles of coupling Porphyrin-Based Sensitizer, 10ml DMF and 50mg targeting biomolecule or antitumor drug were stirring at room reaction 2 hours.
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