CN104162164A

CN104162164A - Indocyanine green-containing polymer albumin nanosphere and preparation method and application thereof

Info

Publication number: CN104162164A
Application number: CN201410337773.9A
Authority: CN
Inventors: 蔡林涛; 盛宗海; 胡德红
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: ZHUHAI INSTITUTE OF ADVANCED TECHNOLOGY CHINESE ACADEMY OF SCIENCES Co.,Ltd.
Priority date: 2013-09-27
Filing date: 2014-07-16
Publication date: 2014-11-26
Anticipated expiration: 2034-07-16
Also published as: CN104162165A; CN104162164B; CN104162171A; CN104162172A; CN103495179A; CN104189916A; CN104162171B; CN104189916B; CN104162172B

Abstract

The invention provides a polymer albumin nanosphere; the polymer albumin nanosphere includes albumin molecules containing thiol and / or disulfide bonds, the albumin molecules are connected with each other by the disulfide bonds, the polymer albumin nanosphere packs a target delivery matter; the target delivery matter includes an anticancer drug and a contrast agent; and the polymer albumin nanosphere diameter is 10-1000nm. The polymer albumin nanosphere is a safe, stable and biocompatible nano carrier capable of packing a drug, or a contrast agent or other target delivery matter, is high in storage stability, and is conducive to long time, effective, and stable release of the target delivery matter; in addition, the polymer albumin nanosphere is small in diameter, uniform in size, and good in dispersion; the invention also provides a preparation method and application of the polymer albumin nanosphere.

Description

A kind of polymer albumin nanospheres that comprises indocyanine-green and its preparation method and application

It is 201310449770.X that the application requires within 27th, to submit the application number of Patent Office of the People's Republic of China in JIUYUE in 2013, its denomination of invention is the priority of the Chinese patent application of " a kind of polymer albumin nanospheres and its preparation method and application ", and its partial content is by reference in conjunction with in this application.

Technical field

The present invention relates to biological medicine Material Field, be specifically related to a kind of polymer albumin nanospheres and its preparation method and application.

Background technology

The rise of nanotechnology makes the drug conveying based on high molecular nanometer microgranule obtain widely paying close attention to, and the various features such as that albumin has is biodegradable, nontoxic, no antigen, are considered to a desirable pharmaceutical carrier.And the size of common single albumin molecule is in several nanometers, be not suitable for being directly used in medicine carrying, supersound method and desolventizing method can make the albumin nanospheres that particle diameter is less than 1 μ m, can be used for delivering medicine, but because the water solublity of albumin molecule is high, the Release Performance of the albumin nanospheres carrier that these class methods make is not easy to control, and how to make albumin nanometer rice grain in water, have good stability, and under diluting condition, not dissolve be the difficult point in current technology of preparing.

The cross-linking agent such as glutaraldehyde are often used to the stable nanosphere obtaining, but the amino sites on glutaraldehyde meeting nonselective albumin-binding surface can discharge aldehydes residue in vivo, and organism is had to remarkable toxic and side effects.Therefore, be necessary to provide a kind of method of preparing safety, albumin nanospheres that stability is high.

Optical therapeutic is a kind of Wicresoft oncotherapy technology that development in recent years is got up, and has been subject to the extensive concern of researcher.At present, optical agents can be divided into the inorganic and large class of organic optical reagent two according to the difference of material.Inorganic optical therapeutic reagent exists residual limitation in difficult degradation, body.Organic optical reagent is easily degraded and is subject to the extensive concern of researcher because having in good biocompatibility, body.

The machine optical agents of common are has chlorin and indocyanine green, chlorin e 6 (chlorin e6, Ce6) as second filial generation sound sensitiser, there is nontoxic, tumor tissues high selectivity and nonneoplastic tissue clearance rate advantages of higher, under illumination and oxygen exist, can produce singlet oxygen and/or free radical and for the photodynamic therapy of tumor.But chlorin e 6 is water insoluble, poor stability, cannot make water solution system, even if first use organic solvent dissolution, be then dispersed in aqueous solution, and be also to exist with suspension form, be difficult to effectively be absorbed by cell; And also there is certain toxicity to cell in organic solvent itself.Indocyanine green (ICG) is a kind of three carbon cyanine dyes with near-infrared characteristic absorption peak, under illumination and oxygen exist, can produce singlet oxygen and/or free radical and for the photodynamic therapy of tumor, can be heat energy and for the photo-thermal therapy of tumor by the luminous energy Partial Conversion of absorption simultaneously.ICG is also current unique nir dye of being ratified clinical use by FDA (Food and Drug Adminstration) (FDA) and Chinese food pharmaceuticals administration general bureau (SFDA), has good biocompatibility, and metabolic pathway is the advantage such as clearly.But as a kind of organic molecule, also there is unstable, easy decomposition, lack the deficiencies such as tumor-targeting in ICG in optical therapeutic process.Therefore, how to improve stability and the targeting of hydrophilic and stability and the ICG of Ce6, making Ce6 and ICG enter quickly and efficiently cell is the focus of studying at present to improve the curative effect of optical therapeutic.

Summary of the invention

For addressing the above problem, the invention provides a kind of polymer albumin nanospheres, this polymer albumin nanospheres comprises containing the albumin molecule of sulfydryl and/or disulfide bond, between described albumin molecule, by disulfide bond, interconnects, and has higher stability under diluting condition; In addition, the particle diameter of this polymer albumin nanospheres is 10～1000nm, and its size homogeneous, and good dispersion is the good carrier that the targets such as delivery medicine or contrast agent are delivered thing, is conducive to discharge and deliver thing for a long time effectively, safely and steadly in patient body; The present invention also provides a kind of preparation method and application of polymer albumin nanospheres.

First aspect, the invention provides a kind of polymer albumin nanospheres, described polymer albumin nanospheres comprises the albumin molecule that contains sulfydryl and/or disulfide bond, between described albumin molecule, by disulfide bond, interconnects, and the particle diameter of described polymer albumin nanospheres is 10～1000nm.

Preferably, the described albumin molecule containing sulfydryl and/or disulfide bond is at least one in human serum albumin, bovine serum albumin, porcine hemoglobin, Recombinant Serum Albumin and hemoglobin.

Preferably, in described polymer albumin nanospheres, bag is loaded with target and delivers thing.

Further preferably, to deliver thing be at least one in cancer therapy drug and contrast agent for described target.

Further preferably, described cancer therapy drug is the coordination compound of platinum and platinum, 5 β, 20-epoxy-1, 2 α, 4, 7 β, 10 β, 13 α-hexahydroxy taxane-11-alkene-9-ketone-4, 10-diacetate esters-2-benzoate-13[(2 ' R, 3 ' S)-N-benzoyl-3-phenylisoserine ester] (paclitaxel), (7S:9S)-9-glycolyl-4-methoxyl group-7, 8, 9, 10-tetrahydrochysene-6, 7, 9, 11-tetrahydroxy-7-0-(2 ', 3 ', 6 ',-tri-deoxidation-3 '-chloros-a-1-lysol is pyranose)-5, 12-naphthalenedione (amycin), (E, E)-1, two (the 4-hydroxy 3-methoxybenzene bases)-1 of 7-, 6-heptadiene-3, 5-diketone (curcumin), 1, 3, 5, 8-tetramethyl-2, 4-bis-(a-ethoxy) porphin phenol-6, 7-dipropionic acid (hemoporphyrin), 4-ethyl-4, 12-dioxy-4-hydroxyl-1H-pyrans (3', 4', 6, 7) pirlindole (1, 2-6) quinoline-3, 14-diketone (camptothecine), (2S-is trans)-18-carboxyl-20-(carboxymethyl)-13-ethyl-2, 3-dihydro 3, 7, 12, 17-tetramethyl-8-vinyl-21H, 23H-porphin-2-propanoic acid (chlorin e 6), IR700 iodide and 11-chloro-1, 1'-diη-propyl-3, 3, 3', 3'-tetramethyl-10, at least one in 12-trimethylene indole three carbon cyanine iodine salt (IR780).

Further preferably, described contrast agent is 2, 7-two [1, 3-dihydro-1, 1-dimethyl-3-(4-sulphur butyl)-1, 3, 5-heptantriene list sodium salt (indocyanine-green), p-[(2, 4-diaminourea pteridine-6)-N-methyl methylamino] benzoyl glutamic acid (methotrexate), 3, two (dimethylamino) phenothiazine-5-father-in-law chlorides (methylene blue) of 7-, 6, 6'-[[3, 3'-dimethyl (1, 1'-diphenyl)-4, 4'-bis-bases] two (azo groups)] two (4-amino-5-hydroxyl-1, 3-naphthalenedisulfonic acid) tetrasodium salt (azovan blue), 2-((4-lignocaine) benzene) (4-(lignocaine) cyclohexane extraction-2, 5-diene) methane) phenyl-1, 4-disulfonate (isosulfan blue), 4, two (lignocaine) triphen dehydration of 4'-methanol-2'', at least one in 4''-disulfonate sodium (patent blue) and metal nanoparticle.

Due to albumin monomer molecule ease of solubility, the albumin nanospheres that physical method is reunited easily disintegrates, the medicine that in nanosphere, bag carries is easy to be discharged prematurely, be unfavorable for the transportation of the target delivery things such as medicine in human recycle system, the controllability of Release Performance that is the physical method albumin nanospheres carrier of reuniting is not high, and polymer albumin nanospheres provided by the invention is interconnected by intermolecular disulfide bond by a plurality of albumin monomer molecules, the stable polymer structure of this chemical bond is more stable than the protein nano ball of assembling by physical method, be difficult for being diluted by human body fluid, dissolve and disintegrate, contribute to the administration concentration that guarantees that targeting moiety is enough.

In biomedical applications, the particle diameter of nanoparticle medicine is important, and different particle diameter metabolic pathway is also different, and small particle diameter is by kidney metabolism, and large particle diameter passes through hepatic metabolism; Wherein, the particle of 20～200nm has passive target effect to tumor, and the nanoparticle medicine in this particle size range can reduce the toxic and side effects of medicine itself, has also increased curative effect simultaneously.

In addition, the dispersibility of nanoparticle medicine is important, and dispersibility is bad, and particle is easily reunited, and even precipitation, causes application comparatively difficulty, particularly its biomedical applications, and dispersibility is even more important.

Little and the size homogeneous of polymer albumin nanospheres particle diameter provided by the invention, good dispersion, with this nanosphere, comprise that delivering medicine has greater advantage in biomedical applications, is conducive to nanosphere and is entered inside tumor and be targeted to tumor cell by gp60 path by EPR effect.

In addition, the molecular weight of general free protein molecule is mostly less than 60KDa, polymer albumin nanospheres provided by the invention is assembled and is formed by a plurality of albumin monomer molecules, molecular weight is higher than 60KDa, be difficult for by glomerular filtration (under the filtration due to glomerule, the protein molecule that general molecular weight is less than 60KDa can be eliminated in the process of metabolism, and can not reach target location), can improve the delivery efficiency that the targets such as medicine are delivered thing.

Second aspect, the invention provides a kind of preparation method of polymer albumin nanospheres, comprises the following steps:

(1) prepare the albumin aqueous solution containing sulfydryl and/or disulfide bond that volume mass concentration is 0.01～300mg/mL, and to regulate the pH value of described albumin aqueous solution be 7～12;

(2) in the albumin aqueous solution that is 7～12 to the described pH value of step (1) gained, add the reducing agent with sulfydryl to obtain reactant liquor, then at 0～60 ℃, shake gently reaction 0.05～12 hour, in described reactant liquor, the molal quantity of the described reducing agent with sulfydryl is 10～5000 times of albumin molal quantity;

(3) the reacted solution of step (2) is carried out ultrasonic under the condition of 0～60 ℃, described ultrasonic power bracket is 1～100KW, simultaneously under the condition stirring, described, with the speed of 0.01～1000ml/s, add organic solvent to obtain microemulsion solution in carrying out ultrasonic solution, described microemulsion solution is reacted after 5～240min under the condition of 0～60 ℃, stratification, then remove organic facies, obtain the suspension containing polymer albumin nanospheres, wherein, the volume that described organic solvent adds is 1～100 times of the reacted solution of described step (2);

(4) by dialysing under the condition that is 7～12 at 0～60 ℃ and pH value containing the suspension of polymer albumin nanospheres of step (3) gained, obtain polymer albumin nanospheres solution;

(5) the polymer albumin nanospheres solution of step (4) gained is carried out to drying and dehydrating processing, obtain polymer albumin nanospheres, described polymer albumin nanospheres comprises the albumin molecule containing sulfydryl and/or disulfide bond, between described albumin molecule, by disulfide bond, interconnect, the particle diameter of described polymer albumin nanospheres is 10～1000nm.

Preferably, in described step (1), the described albumin molecule containing sulfydryl and/or disulfide bond is at least one in human serum albumin, bovine serum albumin, porcine hemoglobin, Recombinant Serum Albumin and hemoglobin.

Preferably, in described step (1), in described albumin aqueous solution, contain volume fraction and be at least one in 0%～20% dimethyl sulfoxide, methanol, ethanol, propanol and the tert-butyl alcohol.

Dimethyl sulfoxide, methanol, ethanol, propanol or the tert-butyl alcohol that the present invention adopts can improve medicine or the dissolubility of contrast agent in solution, and medicine or contrast agent are sufficiently uniformly dissolved.

Preferably, in described step (1), contain target and deliver thing in described albumin aqueous solution, the quality of described target delivery thing is 0.0002～0.5 times of described albumin quality.

Further preferably, at least one in described cancer therapy drug is platinum and platinum analog, paclitaxel, amycin, curcumin, hemoporphyrin, camptothecine, chlorin e 6, IR700 iodide and IR780.

Further preferably, described contrast agent is at least one in indocyanine-green, methotrexate, methylene blue, azovan blue, isosulfan blue, patent blue and metal nanoparticle.

Dimethyl sulfoxide, methanol, ethanol, propanol or the tert-butyl alcohol that the present invention adopts can also improve target and deliver the dissolubility of thing (as medicine or contrast agent) in solution, making target deliver thing fully dissolves and mixs homogeneously with protein, and target is delivered thing and can fully be contacted with albumin molecule, for carrying target delivery thing, albumin bag in next step ultrasonic procedure prepares.

Preferably, in described step (2), the described reducing agent with sulfydryl is glutathion, cysteine, mercaptoethanol or dithiothreitol, DTT.

Preferably, in described step (3), described organic solvent is at least one in methanol, ethanol, propanol and the tert-butyl alcohol.

Further preferably, in described organic solvent, also contain at least one in dimethyl sulfoxide, chloroform, dichloromethane and normal hexane.

Preferably, the described reactant liquor of step (2) shakes gently reaction at 4～60 ℃, in step (3), the reacted solution of step (2) carries out ultrasonic under the condition of 4～60 ℃, described microemulsion solution is reacted 5～240min under the condition of 4～60 ℃, and the described suspension containing polymer albumin nanospheres of step (4) is dialysed at 4～60 ℃.

Preferably, in described step (3), the volume that described organic solvent adds is 2～50 times of the reacted solution of described step (2).

More preferably, in described step (3), the volume that described organic solvent adds is 2～20 times of the reacted solution of described step (2).

Preferably, in described step (4), described method of dialysing is: first by step (3) gained containing the suspension of polymer albumin nanospheres, to be placed in pH value be that 7～12 PBS buffer is dialysed 10～300 hours, then in distilled water, dialyse 12 hours, obtain the solution containing polymer albumin nanospheres.

It is that 7～12 PBS buffer is dialysed that the present invention adopts pH value, can remove on the one hand the impurity such as inorganic molecules in the suspension of polymer albumin nanospheres, on the other hand, under alkali condition, some disulfide bond being present between polymer albumin nanospheres disconnects, then and separately the protein molecule in nanosphere forms new disulfide bond, thereby make the polymer albumin nanospheres separation of assembling, reach the object of disperseing polymer albumin nanospheres, and then form the polymer albumin nanospheres of single dispersion.

As described herein, the polymer albumin nanospheres of described single dispersion is the polymer albumin nanospheres of particle size range between 10～1000nm.Under alkali condition, before dialysis treatment, between some polymer albumin nanospheres, can reunite, cause the particle diameter of the nanosphere after reuniting bigger than normal, after dialysis treatment under alkali condition, the nanosphere of reunion is disperseed, and forms the less nanosphere of particle diameter.

It is that 7～12 buffer is dialysed that the present invention adopts pH value, can remove on the one hand the impurity such as inorganic molecules in the suspension of nano-probe, the more important thing is, the nano-probe of reuniting can be separated into the nanosphere that particle diameter is less under alkali condition, thereby obtains dispersion, the uniform polymer albumin nanospheres of particle diameter.

Preferably, in described step (5), in described polymer albumin nanospheres, bag is loaded with target and delivers thing.

Preferably, the mode that the described polymer albumin nanospheres solution to step (4) gained carries out drying and dehydrating processing is: the polymer albumin nanospheres solution of step (4) gained is placed at 0～-20 ℃ to pre-freeze and after 1～48 hour, is transferred at-20～-80 ℃ freezing 2～48 hours, then lyophilization 12～120 hours in freezer dryer.

The preparation method of polymer albumin nanospheres provided by the invention, the mode that has adopted ultrasonic limit, limit that organic solvent (ethanol or containing the ethanol of the non-polar solven such as chloroform) is injected to ultrasonic emulsification-desolventizing method of albumin solution is prepared nanosphere, on the one hand, when injecting albumin solution, the organic solvents such as ethanol protein molecule can be separated out, meanwhile, formation polymer albumin nanospheres is assembled in the intermolecular formation because of disulfide bond of albumin.On the other hand, control the injection rate of the organic solvents such as the size of ultrasonic power and ethanol well, can control the particle diameter of prepared polymer albumin nanospheres.This be because, when ultrasonic power is large, the injection rate of the organic solvents such as ethanol is large, the particle diameter of the polymer albumin nanospheres of gained is just little; Otherwise when ultrasonic power is little, the injection rate of the organic solvents such as ethanol is little, the particle diameter of the polymer albumin nanospheres of gained is just large.Therefore, the preparation method of polymer albumin nanospheres provided by the invention not only can obtain polymer albumin nanospheres, and can obtain the controlled polymer albumin nanospheres of particle diameter, method provided by the invention can be prepared the polymer albumin nanospheres of particle diameter between 10～1000nm.

In addition, the preparation method of polymer albumin nanospheres provided by the invention, albumin aqueous solution and target are delivered to the first fully mixing of thing, make albumin just deliver thing with target before bag carries target delivery thing and fully contact, can improve the efficiency that albumin bag carries target delivery thing.

The third aspect, the application of the preparation method that the invention provides polymer albumin nanospheres as described in first aspect or the polymer albumin nanospheres as described in second aspect in the medicine of preparation prevention, treatment or cancer diagnosis.

As used herein, " cancer " comprises tumor.

Polymer albumin nanospheres the invention provides and its preparation method and application has following beneficial effect:

(1) polymer albumin nanospheres provided by the invention is interconnected to form nanometer pelletizing by different albumin molecules by disulfide bond between molecule, under water, phosphate buffer, ethanol, serum or culture medium equal solvent diluting condition, than the albumin carrier of physical bond, there is higher stability;

(2) the albumin carrier as stable in glutaraldehyde etc. with using chemical cross-linking agent compared, albumin nanospheres provided by the invention has adopted the disulfide bond of protein molecule itself to obtain stable nanosphere, and therefore albumin nanospheres provided by the invention is safer;

(3) particle diameter of polymer albumin nanospheres provided by the invention is 10～1000nm, and its size homogeneous, good dispersion, is the good carrier that the targets such as delivery medicine or contrast agent are delivered thing, is conducive to discharge and deliver thing for a long time effectively, safely and steadly in patient body;

(4) polymer albumin nanospheres provided by the invention can be used for preparing the medicine of prevention, treatment or cancer diagnosis.

Fourth aspect, the invention provides a kind of polymer albumin nanospheres, and described polymer albumin nanospheres comprises containing the albumin molecule of sulfydryl and/or disulfide bond, between described albumin molecule, by disulfide bond, interconnect,

In described polymer albumin nanospheres, bag is loaded with target and delivers thing;

Described target is delivered thing and is comprised cancer therapy drug and contrast agent;

Described cancer therapy drug comprises (2S-is trans)-18-carboxyl-20-(carboxymethyl)-13-ethyl-2,3-dihydro 3,7,12,17-tetramethyl-8-vinyl-21H, 23H-porphin-2-propanoic acid (chlorin e 6);

Described contrast agent comprises 2,7-two [1,3-dihydro-1,1-dimethyl-3-(4-sulphur butyl)-1,3,5-heptantriene list sodium salts (indocyanine-green);

The particle diameter of described polymer albumin nanospheres is 10～1000nm.

As used herein, " indocyanine-green " claims again indocyanine green.

Preferably, ICG of the present invention is preferably the ICG of the noresidue iodine of medical grade.

As described in the present invention, " polymer albumin nanospheres " is by interconnecting by disulfide bond between albumin molecule, owing to containing at least one sulfydryl or disulfide bond in monomer albumin molecule, if the sulfydryl in this albumin monomer molecule or disulfide bond do not react when forming polymer albumin nanospheres, likely be retained in polymer albumin ball, therefore, polymer albumin nanospheres provided by the invention can contain sulfydryl, disulfide bond, or contain sulfydryl and disulfide bond simultaneously, be that polymer albumin nanospheres provided by the invention can contain sulfydryl and/or disulfide bond.

Preferably, the particle diameter of described polymer albumin nanospheres is 60～200nm.

Preferably, the particle diameter of described polymer albumin nanospheres is 400～500nm.

Preferably, the particle diameter of described polymer albumin nanospheres is 200～300nm.

Preferably, the particle diameter of described polymer albumin nanospheres is 300～400nm.

Preferably, the particle diameter of described polymer albumin nanospheres is 500～700nm.

Preferably, the particle diameter of described polymer albumin nanospheres is 700～1000nm.

As described herein, wrap up target delivery thing and refer to that described polymer albumin is as carrier in described polymer albumin nanospheres, bag carries a described target delivers a thing.

Preferably, the mode that described polymer albumin bag carries target delivery thing is: part target is delivered thing and wrapped up by described albumin molecule, and it is intermolecular that the target delivery thing of remainder is embedded in described albumin.

More preferably, described chlorin e 6 is wrapped up by described albumin molecule, and described indocyanine green part is wrapped up by described albumin molecule, and it is intermolecular that remainder is embedded in described albumin.

Preferably, to deliver the quality of thing be 0.0002～0.5 times of the described albumin quality containing sulfydryl and/or disulfide bond for described target.

Preferably, to deliver the quality of thing be 0.0008～0.5 times of the described albumin quality containing sulfydryl and/or disulfide bond for described target.

Preferably, to deliver the quality of thing be 0.008～0.5 times of the described albumin quality containing sulfydryl and/or disulfide bond for described target.

Albumin is a kind of biological endogenous property albumen, has the advantages such as biodegradable, nontoxic, but albumin can dissolve under diluting condition, unstable in vivo, can not enter target organ by payload drug molecule.

Polymer albumin of the present invention comprises the albumin molecule containing sulfydryl and/or disulfide bond, between described albumin molecule, by disulfide bond, interconnects, and polymer albumin has good stability, can be in aqueous solution stable existence, can be used as good pharmaceutical carrier.

The present invention is loaded in hydrophobicity chlorin e 6 bag in hydrophilic polymer albumin, can improve the water solublity of chlorin e 6 (Ce6), contributes to chlorin e 6 to enter quickly and efficiently cell to improve the curative effect of photodynamic therapy.Polymer albumin nanospheres of the present invention can stable existence in organism inner blood blood circulation, thereby avoid the invalid release of chlorin e 6 in blood circulation transmitting procedure; Meanwhile, due to growth selectivity, high-permeability and the anelasticity (EPR effect) that has increased macromole class material and lipid particle out of control of tumor tissues, for polymer albumin nanospheres provides a kind of ability of passive target tumor tissues; Tumor tissue cell's film rich surface is containing Albumin receptors such as gp60, gp30 and gp18, for polymer albumin nanospheres provides a kind of ability of active target tumor tissue, thereby improved the targeting of polymer albumin nanospheres.

Indocyanine green (ICG) is current unique nir dye of being ratified clinical use by FDA (Food and Drug Adminstration) (FDA) and Chinese food pharmaceuticals administration general bureau (SFDA), in polymer albumin nanospheres provided by the invention, contain ICG, therefore, polymer albumin nanospheres provided by the invention can be as fluorescence living imaging and photoacoustic imaging probe, have good biocompatibility, metabolic pathway is the advantage such as clearly.

Yet as a kind of organic molecule, also there is unstable, easy decomposition, lack the deficiencies such as tumor-targeting in ICG in process of clinical application.

The present invention adopts the stable polymer albumin nanospheres bag of disulfide bond to carry ICG, can avoid invalid release and the decomposition of ICG in blood circulation transmitting procedure, improves the ICG stability in blood circulation in vivo.

In addition, ICG is a kind of three carbon cyanine dyes with near-infrared characteristic absorption peak, under existing, illumination and oxygen can produce singlet oxygen and/or free radical and for the photodynamic therapy of tumor, can be simultaneously heat energy and for the photo-thermal therapy of tumor by the luminous energy Partial Conversion of absorption, therefore, polymer albumin nanospheres provided by the invention also can be used for photodynamics, the photo-thermal therapy of cancer, tumor.

Described polymer albumin nanospheres also has good near-infrared fluorescent and photoacoustic imaging ability, can be used for real-time, the front nanoparticle of noinvasive ground monitor therapy conveying behavior in vivo, after optical dynamic therapy, can to curative effect, carry out real-time assessment by imaging, be embodied as the photodynamic therapy of picture guiding.

Polymer albumin nanospheres particle diameter of the present invention is less, particle favorable dispersibility.

The 5th aspect, the invention provides a kind of preparation method of polymer albumin nanospheres, comprises the following steps:

Wherein, in described albumin aqueous solution, contain target and deliver thing;

Described cancer therapy drug comprises (2S-is trans)-18-carboxyl-20-(carboxymethyl)-13-ethyl-2,3-dihydro 3,7,12,17-tetramethyl-8-vinyl-21H, 23H-porphin-2-propanoic acid;

Described contrast agent comprises 2,7-two [1,3-dihydro-1,1-dimethyl-3-(4-sulphur butyl)-1,3,5-heptantriene list sodium salts;

(5) the polymer albumin nanospheres solution of step (4) gained is carried out to drying and dehydrating processing, obtain polymer albumin nanospheres, described polymer albumin nanospheres comprises the albumin molecule containing sulfydryl and/or disulfide bond, between described albumin molecule, by disulfide bond, interconnect

Wherein, in described polymer albumin nanospheres, bag is loaded with target and delivers thing;

The particle diameter of described polymer albumin nanospheres is 10～1000nm.

Preferably, the mass ratio of described ICG and Ce6 is 1:0.02～50.

Preferably, in described step (1), the quality of described target delivery thing is 0.0002～0.5 times of the described albumin quality containing sulfydryl and/or disulfide bond.

Further preferably, to deliver the quality of thing be 0.0008～0.5 times of the described albumin quality containing sulfydryl and/or disulfide bond for described target.

Further preferably, in described step (1), the quality of described target delivery thing is 0.008～0.5 times of the described albumin quality containing sulfydryl and/or disulfide bond.

Preferably, in described step (2), the molal quantity of the described reducing agent with sulfydryl is 10～100 times of albumin molal quantity.

Preferably, in described step (2), the molal quantity of the described reducing agent with sulfydryl is 100～2500 times of albumin molal quantity.

Preferably, in described step (2), the concentration of the described reducing agent with sulfydryl is 0.01～2mol/L.

Preferably, in described step (3), described ultrasonic power bracket is 50～100KW.

Alternative, described in step of the present invention (3) solution is carried out to ultrasonic object is in order to increase albumin in solution, target, to deliver the dispersibility of thing, and making it abundant contact, this ultrasonic step can adopt other hybrid modes in industry, such as stirring.

Alternative, in described step (3), described reacted microemulsion solution can just can obtain the suspension containing polymer albumin nanospheres without the step of removing organic facies.Stratification is when the organic solvent adopting and water are immiscible, just need to carry out stratification, and get rid of, on the contrary, if the organic solvent that adopts ethanol etc. and water to dissolve each other is not need stratification, the step of removal organic facies.

Further preferably, in described step (3), in described organic solvent, also contain at least one in dimethyl sulfoxide, chloroform, dichloromethane and normal hexane.

Preferably, in described step (3), contain target and deliver thing in described organic solvent, it is chlorin e 6 and ICG that described target is delivered thing.

The present invention can add target to deliver thing in the albumin solution of step (1); Alternative, can also in the organic solvent of step (3), add target to deliver thing; Alternative, can be simultaneously in the albumin solution of step (1), and in the organic solvent of step (3), add target to deliver thing.

While adding chlorin e 6 in the albumin solution of step (1) and/or in step (3) organic solvent, can add again dimethyl sulfoxide (DMSO), oxolane and N, one or more in dinethylformamide, to improve the dissolubility in albumin solution and/or in organic solvent of chlorin e 6.

Preferably, in described step (3), the speed of described organic solvent is incorporated as 50～1000mL/s.

The preparation method of polymer albumin nanospheres provided by the invention adds organic facies (organic solvent) in water (albumin solution), alternative, also water can be added in organic facies.

Preferably, in described step (4), described in the method for dialysing be: it is that 7～12 buffer is dialysed that the suspension containing polymer albumin nanospheres of step (3) gained is placed in to pH value, obtains polymer albumin nanospheres solution.

Further preferably, in described step (4), the described pH for the buffer of dialysing is 9～12.

Further preferably, in described step (4), described is PBS buffer or Tris buffer for the buffer of dialysing.

Further preferably, in described step (4), the dialysis time of the described suspension containing polymer albumin nanospheres in buffer is 10～300 hours.

Further preferably, in described step (4), after being placed in pH value and being 7～12 buffer dialysis containing the suspension of polymer albumin nanospheres, then be placed in dialysis in distilled water.

Still more preferably, being placed in the time of dialysing in distilled water is 1～24 hour.

It is that 7～12 PBS buffer is dialysed that the present invention adopts pH value, can remove containing the impurity such as inorganic molecules in the suspension of polymer albumin nanospheres on the one hand, the more important thing is, that reunites can be separated into the nanosphere that particle diameter is less containing polymer albumin nanospheres under alkali condition, thereby obtains dispersion, the uniform polymer albumin nanospheres of particle diameter.

Polymer albumin nanospheres particle size range provided by the invention is between 10～1000nm, yet, this is not the particle size distribution of the nano-probe of same batch of preparation, on the contrary, adopt the particle size distribution range of nano-probe of each batch that preparation method provided by the invention obtains narrower, such as between 20～250nm, therefore, the size ratio of nano-probe prepared by the present invention is more even, and size is controlled.

Preferably, in described step (5), the mode that the described polymer albumin nanospheres solution to step (4) gained carries out drying and dehydrating processing is that lyophilization, spraying are dried or distilling under reduced pressure.

Further preferably, in described step (5), described cryodesiccated step is: the polymer albumin nanospheres solution of step (4) gained is placed at 0～-20 ℃ to pre-freeze and after 1～48 hour, is transferred at-20～-80 ℃ freezing 2～48 hours, then lyophilization 12～120 hours in freezer dryer.

The present invention contains sulfydryl and/or disulfide bond albumin by control and with the mol ratio of the reducing agent of sulfydryl, mol ratio containing sulfydryl and/or disulfide bond albumin and target delivery thing, the pH of each step, especially the speed that the pH of dialysis buffer liquid, and organic facies and water mix has obtained the polymer albumin nanospheres that particle diameter is little, particle size distribution range is narrower, particle diameter is controlled.

The polymer albumin nanospheres that fifth aspect present invention makes has following beneficial effect:

(1) noresidue: adopting polymer albumin is carrier, and ICG and Ce6 are optical agents, three all has the advantages such as safety non-toxic, non-immunogenicity, biodegradable, good biocompatibility.Polymer albumin is intermolecular to be connected with disulfide bond, this particle can stable existence in organism inner blood blood circulation, thereby avoid ICG and the Ce6 invalid release in blood circulation transmitting procedure, and under the effect of reduced glutathion (GSH), degrade and discharge monomer albumin, ICG and Ce6 in cell, albumin is degraded by natural metabolism in vivo, thereby can not introduce in vivo any exogenous material;

(2) targeting is good: on the one hand, growth selectivity, high-permeability and the anelasticity (EPR effect) that has increased macromole class material and lipid particle out of control of tumor tissues, for polymer albumin nanospheres provides a kind of ability of passive target tumor tissues; On the other hand, tumor tissue cell's film rich surface is containing Albumin receptors such as gp60, gp30, gp18, for polymer albumin nanospheres provides a kind of ability of active target tumor tissue; On the other hand, utilize the acute inflammation producing after Ce6 optical dynamic treatment of tumor can impel polymer albumin nanospheres in the further enrichment of tumor locus.Three's combination, has improved the targeting of polymer albumin nanospheres;

(3) visual: polymer albumin nanospheres has good near-infrared fluorescent and photoacoustic imaging ability simultaneously, can be used for monitoring conveying behavior in the front nanoparticle daughter of optical therapeutic in real time, noinvasive, after optical therapeutic, can to curative effect, carry out real-time assessment by imaging, be embodied as the optical therapeutic of picture guiding.

Preparation method of the present invention is simple, and reaction condition is gentle, and reaction repeatability is good, and the polymer albumin nanospheres particle diameter making is little, good dispersion.

The 6th aspect, the application of the preparation method that the invention provides polymer albumin nanospheres as described in fourth aspect or the polymer albumin nanospheres as described in the 5th aspect in the medicine of preparation prevention, treatment or cancer diagnosis.

Preferably, the application of the preparation method that is applied as polymer albumin nanospheres or polymer albumin nanospheres described in preparing photodynamic tumor medicine, tumor photo-thermal therapy medicine, fluorescence imaging probe and photoacoustic imaging probe.

As used herein, " cancer " comprises tumor.

The application of polymer albumin nanospheres of the present invention has following beneficial effect: utilize the acute inflammation producing after Ce6 optical dynamic treatment of tumor can impel polymer albumin nanospheres in the further enrichment of tumor locus, contribute to ICG performance photo-thermal and OPK treatment simultaneously, greatly suppressed the growth of tumor.

Accompanying drawing explanation

Fig. 1 is the scanning electron microscope image of the polymer albumin nanospheres that makes of the embodiment of the present invention one;

Fig. 2 is the images of transmissive electron microscope of the polymer albumin nanospheres that makes of the embodiment of the present invention one;

Fig. 3 is the dilution experiment of the polymer albumin nanospheres that makes of the embodiment of the present invention one in different solvents;

Fig. 4 is polymer albumin nanospheres dissolution experiment under reduced form condition that the embodiment of the present invention one makes;

Fig. 5 is the scanning electron microscope image of the polymer albumin nanospheres that makes of the embodiment of the present invention eight;

Fig. 6 is that the polymer albumin nanospheres that the embodiment of the present invention eight makes is injected into the fluorescence imaging figure after tumor bearing nude mice;

Fig. 7 is that the polymer albumin nanospheres that the embodiment of the present invention eight makes is injected into the photoacoustic imaging figure after tumor bearing nude mice;

Fig. 8 is that the polymer albumin nanospheres that makes of the embodiment of the present invention nine is for the tumor growth curve of tumor bearing nude mice light dynamic experiment.

The specific embodiment

The following stated is the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.

Embodiment mono-

A preparation method for polymer albumin nanospheres, comprises the following steps:

(1) getting 1mL volume mass concentration is 0.01 (w/v, the dimethyl sulphoxide solution of indocyanine-green mg/mL) and 1mL volume mass concentration are 0.02 (w/v, mg/mL) bovine serum albumin mixes, obtain albumin mixed liquor, then adopt the NaOH solution of 0.1mol/L to regulate the pH value to 7 of described albumin mixed liquor;

(2) in the albumin mixed liquor that is 7 to the described pH value of step (1) gained, add glutathion to obtain reactant liquor, then at 0 ℃, shake gently reaction 1 hour, in described reactant liquor, the molal quantity of described glutathion is 10 times of albumin molal quantity;

(3) the reacted solution of step (2) is carried out ultrasonic under the condition of 0 ℃, ultrasonic power is 100KW, simultaneously at the described 4mL dehydrated alcohol injecting with the speed of 1000mL/s in carrying out ultrasonic solution, obtain microemulsion solution, described microemulsion solution is reacted after 5min under the condition of 0 ℃, obtains the suspension containing polymer albumin nanospheres;

(4) suspension containing polymer albumin nanospheres of step (3) gained is moved into bag filter, keeping temperature is under the condition of 0 ℃, bag filter is placed in to 5L pH dialyses 10 hours in 10 PBS buffer, within every 12 hours during this time, change liquid 1 time, each 5L pH that adopts is 10 PBS buffer, and then bag filter is placed in 5L distilled water and is dialysed 1 hour, obtain polymer albumin nanospheres solution;

(5) the polymer albumin nanospheres solution of step (4) gained is placed at negative 20 ℃ to pre-freeze and after 2 hours, is transferred at negative 80 ℃ freezing 12 hours, then lyophilization 12 hours in freezer dryer, obtain polymer albumin nanospheres, described polymer albumin nanospheres comprises the albumin molecule containing sulfydryl and/or disulfide bond, between described albumin molecule, by disulfide bond, interconnect, the particle diameter of described polymer albumin nanospheres is 10～100nm.

For absolutely proving the beneficial effect of polymer albumin nanospheres prepared by the embodiment of the present invention, the present embodiment also provides the scanning electron microscope image of this polymer albumin nanospheres, as shown in Figure 1, Fig. 1 is the scanning electron microscope image of the polymer albumin nanospheres that makes of the embodiment of the present invention one, from this image, polymer albumin nanospheres size homogeneous prepared by the present embodiment, granule comparatively disperses.

The present embodiment also provides transmission electron microscope (TEM) image of this polymer albumin nanospheres, as shown in Figure 2, Fig. 2 is the images of transmissive electron microscope of the polymer albumin nanospheres that makes of the embodiment of the present invention one, from this image, the particle diameter of polymer albumin nanospheres prepared by the present embodiment is 10～100nm, size homogeneous, granule comparatively disperses.

In addition, the present embodiment also provides this dilution experiment of polymer albumin nanospheres in different solvents, concrete operations are: this polymer albumin nanospheres solution is dissolved in respectively in phosphate buffer (pH is 7.4), serum (pH is 7.4) and cell culture medium (pH is 7.4), at different point in time sampling, observe subsequently the particle diameter of this polymer albumin nanospheres, result as shown in Figure 3.

Fig. 3 is the dilution experiment of the polymer albumin nanospheres that makes of the embodiment of the present invention one in different solvents, as shown in Figure 3: the particle diameter temporal evolution of this polymer albumin nanospheres in phosphate buffer, serum and cell culture medium is not obvious, be polymer albumin nanospheres provided by the invention can the dilution experiment under approaching physiological condition in stable existence, there is medical application prospect.

Secondly, the present embodiment also provides the dissolution experiment of this polymer albumin nanospheres under reduced form condition, concrete operations are: this polymer albumin nanospheres solution is dissolved in respectively to the dithiothreitol, DTT that concentration is 20mM, 2 hours, after 8 hours and 24 hours, run respectively SDS-PAGE electrophoresis, detect the dissolution law of this polymer albumin nanospheres under reduced form condition, wherein, a, b, the corresponding polymer albumin nanospheres of c swimming lane difference is at reductase 12 hour, 8 hours, sample after 24 hours, d swimming lane is the contrast of albumin monomer molecule, result as shown in Figure 4.

Fig. 4 is polymer albumin nanospheres dissolution experiment under reduced form condition that the embodiment of the present invention one makes, as shown in Figure 4, band in frame 1 is polymer albumin nanospheres prepared by the present embodiment, band in frame 2 is for forming the band of the albumin oligomer of this polymer albumin nanospheres, band in frame 3 is for forming albumin dimer and the trimerical band of this polymer albumin nanospheres, and the band in frame 4 is for forming the monomolecular band of albumin of this polymer albumin nanospheres; This result shows, the band concentration in the frame 3 that albumin dimer and trimer are corresponding raises with the prolongation of recovery time, and in addition, the band concentration in frame 4 corresponding to protein monomers is also significantly improved with the prolongation of recovery time; Be that this polymer albumin nanospheres that the present embodiment provides can dissolve under the condition of reducing agent existence, and with the prolongation of recovery time, the degree that its disulfide bond is reduced is higher, therefore, when adopting this polymer albumin nanospheres to carry out drug delivery, can be degraded by the reducing substanceses such as reductive glutathione in cell, thereby discharge medicine.

Embodiment bis-

(1) getting 0.1mL volume mass concentration is 0.1 (w/v, mg/mL) dimethyl sulphoxide solution of amycin and 2mL volume mass concentration are 300 (w/v, mg/mL) porcine hemoglobin is mixed, obtain albumin mixed liquor, then adopt the NaOH solution of 1mol/L to regulate the pH value to 7 of described albumin mixed liquor;

(2) in the albumin mixed liquor that is 7 to the described pH value of step (1) gained, add dithiothreitol, DTT to obtain reactant liquor, then at 60 ℃, shake gently reaction 0.05 hour, in described reactant liquor, the molal quantity of described dithiothreitol, DTT is 5000 times of albumin molal quantity;

(3) the reacted solution of step (2) is carried out ultrasonic under the condition of 60 ℃, ultrasonic power is 1KW, the 200mL simultaneously injecting in described speed of take 0.01ml/s in carrying out ultrasonic solution obtains microemulsion solution containing the alcoholic solution (volume ratio of chloroform and ethanol is 1:9) of chloroform, described microemulsion solution is reacted after 20min under the condition of 60 ℃, standingly after microemulsion solution layering, remove organic facies, obtain the suspension containing polymer albumin nanospheres;

(4) suspension containing polymer albumin nanospheres of step (3) gained is moved into bag filter, keeping temperature is under the condition of 30 ℃, bag filter is placed in to 1L pH dialyses 300 hours in 7 PBS buffer, within every 12 hours during this time, change liquid 1 time, each 1L pH that adopts is 7 PBS buffer, and then bag filter is placed in 5L distilled water and is dialysed 24 hours, obtain polymer albumin nanospheres solution;

(5) the polymer albumin nanospheres solution of step (4) gained is placed at 0 ℃ to pre-freeze and after 1 hour, is transferred at negative 20 ℃ freezing 2 hours, then lyophilization 72 hours in freezer dryer, obtain polymer albumin nanospheres, described polymer albumin nanospheres comprises the albumin molecule containing sulfydryl and/or disulfide bond, between described albumin molecule, by disulfide bond, interconnect, the particle diameter of described polymer albumin nanospheres is 900～1000nm.

Embodiment tri-

(1) getting 0.5mL volume mass concentration is 0.5 (w/v, mg/mL) aqueous solution of U.S. basket and 1.5mL200 (w/v, mg/mL) Recombinant Serum Albumin is mixed, and obtains albumin mixed liquor, then adopts the NaOH solution of 2mol/L to regulate the pH value to 12 of described albumin mixed liquor;

(2) in the albumin mixed liquor that is 12 to the described pH value of step (1) gained, add beta-mercaptoethanol to obtain reactant liquor, then at 30 ℃, shake gently reaction 12 hours, in described reactant liquor, the molal quantity of described beta-mercaptoethanol is 100 times of albumin molal quantity;

(3) the reacted solution of step (2) is carried out ultrasonic under the condition of 30 ℃, ultrasonic power is 100KW, the 100mL simultaneously injecting in described speed of take 1000ml/s in carrying out ultrasonic solution obtains microemulsion solution containing the alcoholic solution (volume ratio of chloroform and ethanol is 1:9) of chloroform, described microemulsion solution is reacted after 240min under the condition of 30 ℃, standingly after microemulsion solution layering, remove organic facies, obtain the suspension containing polymer albumin nanospheres;

(4) suspension containing polymer albumin nanospheres of step (3) gained is moved into bag filter, keeping temperature is under the condition of 60 ℃, bag filter is placed in to 1L pH dialyses 144 hours in 12 PBS buffer, each 1L pH that adopts is 12 PBS buffer, within every 12 hours during this time, change liquid 1 time, and then bag filter is placed in 5L distilled water and is dialysed 12 hours, obtain polymer albumin nanospheres solution;

(5) the polymer albumin nanospheres solution of step (4) gained is placed at negative 4 ℃ to pre-freeze and after 48 hours, is transferred at negative 50 ℃ freezing 48 hours, then lyophilization 96 hours in freezer dryer, obtain polymer albumin nanospheres, described polymer albumin nanospheres comprises the albumin molecule containing sulfydryl and/or disulfide bond, between described albumin molecule, by disulfide bond, interconnect, the particle diameter of described polymer albumin nanospheres is 100～200nm.

Embodiment tetra-

(1) getting 1mL volume mass concentration is 1 (w/v, mg/mL) dimethyl sulphoxide solution of chlorin e 6 solution and 1mL300 (w/v, mg/mL) hemoglobin solutions mixes, obtain albumin mixed liquor, the pH value to 9 that then adopts the NaOH solution of 0.5mol/L to regulate described albumin to mix;

(2) in the albumin mixed liquor that is 9 to the described pH value of step (1) gained, add glutathion to obtain reactant liquor, then at 60 ℃, shake gently reaction 0.05 hour, in described reactant liquor, the molal quantity of described glutathion is 2500 times of albumin molal quantity;

(3) the reacted solution of step (2) is carried out ultrasonic under the condition of 60 ℃, ultrasonic power is 10KW, simultaneously at the described 22mL alcoholic solution injecting with the speed of 50ml/s in carrying out ultrasonic solution, obtain microemulsion solution, described microemulsion solution is reacted after 30min under the condition of 60 ℃, obtains the suspension containing polymer albumin nanospheres;

(4) suspension containing polymer albumin nanospheres of step (3) gained is moved into bag filter, keeping temperature is under the condition of 60 ℃, bag filter is placed in to 1L pH dialyses 36 hours in 9 PBS buffer, within every 12 hours during this time, change liquid 1 time, each 1L pH that adopts is 9 PBS buffer, and then bag filter is placed in 5L distilled water and is dialysed 18 hours, obtain polymer albumin nanospheres solution;

(5) the polymer albumin nanospheres solution of step (4) gained is placed at negative 10 ℃ to pre-freeze and after 24 hours, is transferred at negative 80 ℃ freezing 24 hours, then lyophilization 120 hours in freezer dryer, obtain polymer albumin nanospheres, described polymer albumin nanospheres comprises the albumin molecule containing sulfydryl and/or disulfide bond, between described albumin molecule, by disulfide bond, interconnect, the particle diameter of described polymer albumin nanospheres is 600～700nm.

Embodiment five

(1) get the porcine hemoglobin solution that 2mL volume mass concentration is 0.01 (w/v, mg/mL), then adopt the NaOH solution of 1mol/L to regulate the pH value to 7 of described albumin mixed liquor;

(2) in the albumin mixed liquor that is 7 to the described pH value of step (1) gained, add cysteine to obtain reactant liquor, then at 60 ℃, shake gently reaction 0.05 hour, in described reactant liquor, the molal quantity of described cysteine is 10 times of albumin molal quantity;

(3) the reacted solution of step (2) is carried out ultrasonic under the condition of 60 ℃, ultrasonic power is 1KW, simultaneously at the described 4mL alcoholic solution injecting with the speed of 50ml/s in carrying out ultrasonic solution, obtain microemulsion solution, described microemulsion solution is reacted after 20min under the condition of 60 ℃, obtains the suspension containing polymer albumin nanospheres;

(4) suspension containing polymer albumin nanospheres of step (3) gained is moved into bag filter, keeping temperature is under the condition of 30 ℃, bag filter is placed in to 1L pH dialyses 10 hours in 7 PBS buffer, within every 12 hours during this time, change liquid 1 time, each 1L pH that adopts is 7 PBS buffer, and then bag filter is placed in 5L distilled water and is dialysed 1 hour, obtain polymer albumin nanospheres solution;

(5) the polymer albumin nanospheres solution of step (4) gained is placed at 0 ℃ to pre-freeze and after 1 hour, is transferred at negative 20 ℃ freezing 2 hours, then lyophilization 12 hours in freezer dryer, obtain polymer albumin nanospheres, described polymer albumin nanospheres comprises the albumin molecule containing sulfydryl and/or disulfide bond, between described albumin molecule, by disulfide bond, interconnect, the particle diameter of described polymer albumin nanospheres is 700～800nm.

Embodiment six

(1) get the Recombinant Serum Albumin solution of 2mL300 (w/v, mg/mL), then adopt the NaOH solution of 2mol/L to regulate the pH value to 12 of described albumin mixed liquor;

(2) in the albumin mixed liquor that is 12 to the described pH value of step (1) gained, add dithiothreitol, DTT to obtain reactant liquor, then at 30 ℃, shake gently reaction 12 hours, in described reactant liquor, the molal quantity of described dithiothreitol, DTT is 5000 times of albumin molal quantity;

(3) the reacted solution of step (2) is carried out ultrasonic under the condition of 30 ℃, ultrasonic power is 100KW, simultaneously at the described 200mL alcoholic solution injecting with the speed of 1000ml/s in carrying out ultrasonic solution, obtain microemulsion solution, described microemulsion solution is reacted after 240min under the condition of 30 ℃, obtains the suspension containing polymer albumin nanospheres;

(4) suspension containing polymer albumin nanospheres of step (3) gained is moved into bag filter, keeping temperature is under the condition of 60 ℃, bag filter is placed in to 1L pH dialyses 300 hours in 12 PBS buffer, each 1L pH that adopts is 12 PBS buffer, within every 12 hours during this time, change liquid 1 time, and then bag filter is placed in 5L distilled water and is dialysed 24 hours, obtain polymer albumin nanospheres solution;

(5) the polymer albumin nanospheres solution of step (4) gained is placed at negative 4 ℃ to pre-freeze and after 48 hours, is transferred at negative 80 ℃ freezing 36 hours, then lyophilization 96 hours in freezer dryer, obtain polymer albumin nanospheres, described polymer albumin nanospheres comprises the albumin molecule containing sulfydryl and/or disulfide bond, between described albumin molecule, by disulfide bond, interconnect, the particle diameter of described polymer albumin nanospheres is 10～100nm.

Embodiment seven

(1) get the albumin solution of 2mL150 (w/v, mg/mL), then adopt the NaOH solution of 2mol/L to regulate the pH value to 9 of described albumin mixed liquor;

(2) in the albumin mixed liquor that is 9 to the described pH value of step (1) gained, add beta-mercaptoethanol to obtain reactant liquor, then at 0 ℃, shake gently reaction 17 hours, in described reactant liquor, the molal quantity of described beta-mercaptoethanol is 2500 times of albumin molal quantity;

(3) the reacted solution of step (2) is carried out ultrasonic under the condition of 0 ℃, ultrasonic power is 10KW, simultaneously at the described 100mL alcoholic solution injecting with the speed of 0.01ml/s in carrying out ultrasonic solution, obtain microemulsion solution, described microemulsion solution is reacted after 5min under the condition of 0 ℃, obtains the suspension containing polymer albumin nanospheres;

(4) suspension containing polymer albumin nanospheres of step (3) gained is moved into bag filter, keeping temperature is under the condition of 0 ℃, bag filter is placed in to 1L pH dialyses 144 hours in 9 PBS buffer, each 1L pH that adopts is 12 PBS buffer, within every 12 hours during this time, change liquid 1 time, and then bag filter is placed in 5L distilled water and is dialysed 12 hours, obtain polymer albumin nanospheres solution;

(5) the polymer albumin nanospheres solution of step (4) gained is placed at negative 20 ℃ to pre-freeze and after 24 hours, is transferred at negative 50 ℃ freezing 48 hours, then lyophilization 120 hours in freezer dryer, obtain polymer albumin nanospheres, described polymer albumin nanospheres comprises the albumin molecule containing sulfydryl and/or disulfide bond, between described albumin molecule, by disulfide bond, interconnect, the particle diameter of described polymer albumin nanospheres is 700～900nm.

Comparative example one

(1) get the bovine serum albumin solution of 2mL0.01 (w/v, mg/mL);

(2) in the Bovine Serum Albumin in Aqueous Solution of step (1) gained, add cysteine to obtain reactant liquor, then at 60 ℃, shake gently reaction 0.05 hour, in described reactant liquor, the molal quantity of described cysteine is 10 times of albumin molal quantity;

(3) in the solution of described step (2) gained, add 10mL ethanol solution to obtain microemulsion solution, described microemulsion solution is reacted after 10min under the condition of 0 ℃, obtains the suspension containing polymer albumin nanospheres;

(4) by step (3) gained containing the suspension of polymer albumin nanospheres, move into bag filter, keeping temperature is, under the condition of 25 ℃, bag filter to be placed in 5L distilled water and to be dialysed 12 hours, obtains polymer albumin nanospheres solution;

(5) the polymer albumin nanospheres solution of step (4) gained is placed at negative 20 ℃ to pre-freeze and after 2 hours, is transferred at negative 80 ℃ freezing 24 hours, then lyophilization 48 hours in freezer dryer, obtain polymer albumin nanospheres, described polymer albumin nanospheres comprises the albumin molecule containing sulfydryl and/or disulfide bond, between described albumin molecule, by disulfide bond, interconnect, the particle diameter of described polymer albumin nanospheres is 10～600nm.

Comparative example two

(1) getting 1mL volume mass concentration is that the indocyanine-green of 1 (w/v, mg/mL) and the bovine serum albumin of 1mL300 (w/v, mg/mL) mix, and obtains albumin mixed liquor;

(2) in the albumin mixed liquor of step (1) gained, add glutathion to obtain reactant liquor, then at 60 ℃, shake gently reaction 0.05 hour, in described reactant liquor, the molal quantity of described glutathion is 5000 times of albumin molal quantity;

(5) the polymer albumin nanospheres solution of step (4) gained is placed at negative 20 ℃ to pre-freeze and after 2 hours, is transferred at negative 80 ℃ freezing 24 hours, then lyophilization 48 hours in freezer dryer, obtain polymer albumin nanospheres, described polymer albumin nanospheres comprises the albumin molecule containing sulfydryl and/or disulfide bond, between described albumin molecule, by disulfide bond, interconnect, the particle diameter of described polymer albumin nanospheres is 100～1000nm.

The polymer albumin nanospheres providing with respect to the embodiment of the present invention, the polymer albumin nanospheres centralized particle diameter degree that comparative example 1 and comparative example 2 provide is not high, is unfavorable for its application in biomedicine.

Embodiment eight

(1) get the Bovine Serum Albumin in Aqueous Solution that volume mass concentration is 50mg/mL (w/v), then adopt the NaOH solution of 0.1mol/L to regulate the pH value to 7 of described albumin aqueous solution;

(2) in the albumin aqueous solution that is 7 to the described pH value of step (1) gained, add glutathion to obtain reactant liquor, then at 4 ℃, shake gently reaction 2 hours, in described reactant liquor, the molal quantity of described glutathion is 10 times of albumin molal quantity;

(3) the reacted solution of step (2) is carried out ultrasonic under the condition of 4 ℃, ultrasonic power is 100KW, simultaneously described, with the speed of 1000mL/s, inject alcoholic solution in carrying out ultrasonic solution, the ICG that the chlorin e 6 that is 1mg/mL containing dimethyl sulfoxide, concentration in this alcoholic solution and concentration are 0.1mg/mL, in the volume of dimethyl sulfoxide and alcoholic solution, the volume ratio of etoh solvent is 1:9, obtain microemulsion solution, this microemulsion solution obtains the suspension containing polymer albumin nanospheres react 10min under the condition of 4 ℃ after; The volume that alcoholic solution adds is 2 times of the reacted liquor capacity of described step (2);

(4) suspension containing polymer albumin nanospheres of step (3) gained is moved into bag filter, keeping temperature is under the condition of 4 ℃, bag filter is placed in to 1L pH dialyses 24 hours in 9 PBS buffer, within every 8 hours during this time, change liquid 1 time, each 1L pH that adopts is 9 PBS buffer, and then bag filter is placed in 1L distilled water and is dialysed 12 hours, obtain polymer albumin nanospheres solution;

(5) the polymer albumin nanospheres solution of step (4) gained is placed at negative 20 ℃ to pre-freeze and after 2 hours, is transferred at negative 80 ℃ freezing 24 hours, then lyophilization 48 hours in freezer dryer, obtain polymer albumin nanospheres, in described polymer albumin nanospheres, bag is loaded with target and delivers thing; Described target is delivered thing and is comprised chlorin e 6 and ICG, and polymer albumin comprises the albumin molecule that contains sulfydryl and/or disulfide bond, between albumin, by disulfide bond, interconnects, and the particle diameter of polymer albumin nanospheres is 60～200nm.

Fig. 5 is the scanning electron microscope (SEM) photograph of the resulting polymer albumin nanospheres of embodiment 8, and as can be seen from the figure, the particle size range of polymer albumin nanospheres is 60nm～200nm, the less and nanoparticle favorable dispersibility of particle diameter.

Embodiment nine

(1) get the porcine hemoglobin aqueous solution that concentration is 200mg/mL (w/v), then adopt the NaOH solution of 10mol/L to regulate the pH value to 12 of described albumin mixed liquor;

(2) in the albumin mixed liquor that is 12 to the described pH value of step (1) gained, add dithiothreitol, DTT to obtain reactant liquor, then at 60 ℃, shake gently reaction 240 hours, in described reactant liquor, the molal quantity of described dithiothreitol, DTT is 5000 times of albumin molal quantity;

(3) the reacted solution of step (2) is carried out ultrasonic under the condition of 60 ℃, ultrasonic power is 1KW, simultaneously described, with the speed of 1000ml/s, inject alcoholic solution in carrying out ultrasonic solution, the ICG that the chlorin e 6 that is 2.5mg/mL containing dimethyl sulfoxide, volume mass concentration in alcoholic solution and volume mass concentration are 5mg/mL, the volume ratio of the etoh solvent in dimethyl sulfoxide and alcoholic solution is 1:9, obtain microemulsion solution, described microemulsion solution is reacted after 5min under the condition of 60 ℃, obtains the suspension containing polymer albumin nanospheres; The volume that alcoholic solution adds is 2 times of the reacted liquor capacity of described step (2);

(4) suspension containing polymer albumin nanospheres of step (3) gained is moved into bag filter, under room temperature, bag filter is placed in to 5L pH dialyses 300 hours in 12 PBS buffer, within every 12 hours during this time, change liquid 1 time, each 5L pH that adopts is 12 PBS buffer, and then bag filter is placed in 5L distilled water and is dialysed 12 hours, obtain polymer albumin nanospheres solution;

(5) the polymer albumin nanospheres solution of step (4) gained is placed at 0 ℃ to pre-freeze and after 1 hour, is transferred at negative 20 ℃ freezing 2 hours, then lyophilization 72 hours in freezer dryer, obtain polymer albumin nanospheres, in described polymer albumin nanospheres, bag is loaded with target and delivers thing; Described target is delivered thing and is comprised chlorin e 6 and ICG, and polymer albumin comprises the albumin molecule that contains sulfydryl and/or disulfide bond, between albumin molecule, by disulfide bond, interconnects, and the particle diameter of polymer albumin nanospheres is 400～500nm.

Embodiment ten

(1) get albumin mixed liquor, albumin mixed liquor contains the ICG that porcine hemoglobin that concentration is 100mg/mL (w/v) and concentration are 5mg/mL, then adopts the NaOH solution of 0.1mol/L to regulate the pH value to 9 of described albumin aqueous solution;

(2) in the albumin mixed liquor that is 9 to the described pH value of step (1) gained, add beta-mercaptoethanol to obtain reactant liquor, then at 30 ℃, shake gently reaction 0.05 hour, in described reactant liquor, the molal quantity of described beta-mercaptoethanol is 100 times of albumin molal quantity;

(3) the reacted solution of step (2) is carried out ultrasonic under the condition of 30 ℃, ultrasonic power is 50KW, simultaneously described, with the speed of 0.01ml/s, inject alcoholic solution in carrying out ultrasonic solution, in alcoholic solution, contain the chlorin e 6 that dimethyl sulfoxide and volume mass concentration are 0.1mg/mL, the volume ratio of the etoh solvent in dimethyl sulfoxide and alcoholic solution is 1:9, obtain microemulsion solution, described microemulsion solution is reacted after 240min under the condition of 30 ℃, obtains the suspension containing polymer albumin nanospheres; The volume that alcoholic solution adds is 50 times of the reacted liquor capacity of step (2);

(4) suspension containing polymer albumin nanospheres of step (3) gained is moved into bag filter, keeping temperature is under the condition of 60 ℃, bag filter is placed in to 100mL pH dialyses 2 hours in 7 PBS buffer, each 100mL pH that adopts is 7 PBS buffer, within every 1 hour during this time, change liquid 1 time, and then bag filter is placed in 100mL distilled water and is dialysed 12 hours, obtain polymer albumin nanospheres solution;

(5) the polymer albumin nanospheres solution of step (4) gained is placed at negative 4 ℃ to pre-freeze and after 48 hours, is transferred at negative 50 ℃ freezing 48 hours, then lyophilization 96 hours in freezer dryer, obtain polymer albumin nanospheres, in described polymer albumin nanospheres, bag is loaded with target and delivers thing; Described target is delivered thing and is comprised chlorin e 6 and ICG, and polymer albumin comprises the albumin molecule that contains sulfydryl and/or disulfide bond, between albumin molecule, by disulfide bond, interconnects, and the particle diameter of polymer albumin nanospheres is 200～300nm.

Embodiment 11

(1) get albumin mixed liquor, albumin mixed liquor contains the porcine hemoglobin that concentration is 100mg/mL (w/v), the ICG that concentration is 0.1mg/mL, chlorin and the dimethyl sulfoxide that concentration is 5mg/mL, in dimethyl sulfoxide and albumin mixed liquor, the volume ratio of aqueous solvent is 0.1:1, then adopts the pH value to 9 of the NaOH solution adjusting mixed liquor of 0.1mol/L;

(2) in step (1) mixed liquor, add glutathion to obtain reactant liquor, then at 30 ℃, shake gently reaction 1 hour, in reactant liquor, the molal quantity of glutathion is 10 times of albumin molal quantity;

(3) the reacted solution of step (2) is carried out ultrasonic under the condition of 4 ℃, ultrasonic power is 100KW, by carrying out the speed with 100mL/s in ultrasonic solution, be injected in dehydrated alcohol simultaneously, obtain microemulsion solution, described microemulsion solution is reacted after 10min under the condition of 4 ℃, obtains the suspension containing polymer albumin nanospheres; The volume that dehydrated alcohol adds is 50 times of the reacted liquor capacity of step (2);

(4) suspension containing polymer albumin nanospheres of step (3) gained is moved into bag filter, keeping temperature is under the condition of 0 ℃, bag filter is placed in to 100mL pH dialyses 2 hours in 7 PBS buffer, within every 1 hour during this time, change liquid 1 time, each 100mL pH that adopts is 7 PBS buffer, and then bag filter is placed in 100mL distilled water and is dialysed 12 hours, obtain polymer albumin nanospheres solution;

(5) the polymer albumin nanospheres solution of step (4) gained is placed at negative 20 ℃ to pre-freeze and after 2 hours, is transferred at negative 80 ℃ freezing 24 hours, then lyophilization 48 hours in freezer dryer, obtain polymer albumin nanospheres, in described polymer albumin nanospheres, bag is loaded with target and delivers thing; Described target is delivered thing and is comprised chlorin e 6 and ICG, and polymer albumin comprises the albumin molecule that contains sulfydryl and/or disulfide bond, between albumin molecule, by disulfide bond, interconnects, and the particle diameter of polymer albumin nanospheres is 100～200nm.

Embodiment 12

(1) get albumin mixed liquor, albumin mixed liquor contains the porcine hemoglobin that concentration is 100mg/mL (w/v), chlorin and the dimethyl sulfoxide that concentration is 1mg/mL, in dimethyl sulfoxide and albumin mixed liquor, the volume ratio of aqueous solvent is 0.1:1, then adopts the pH value to 9 of the NaOH solution adjusting mixed liquor of 0.1mol/L;

(2) in step (1) gained mixed liquor, add glutathion to obtain reactant liquor, then at 30 ℃, shake gently reaction 1 hour, in described reactant liquor, the molal quantity of described glutathion is 10 times of albumin molal quantity;

(3) the reacted solution of step (2) is carried out ultrasonic under the condition of 0 ℃, ultrasonic power is 50KW, by carrying out the speed with 0.01mL/s in ultrasonic solution, be injected in alcoholic solution simultaneously, the ICG that this alcoholic solution is 1mg/mL containing volume mass concentration, obtain microemulsion solution, described microemulsion solution is reacted after 10min under the condition of 0 ℃, obtains the suspension containing polymer albumin nanospheres; The volume that alcoholic solution adds is 20 times of the reacted liquor capacity of step (2);

(5) the polymer albumin nanospheres solution of step (4) gained is placed at negative 20 ℃ to pre-freeze and after 2 hours, is transferred at negative 80 ℃ freezing 24 hours, then lyophilization 48 hours in freezer dryer, obtain polymer albumin nanospheres, in described polymer albumin nanospheres, bag is loaded with target and delivers thing; Described target is delivered thing and is comprised chlorin e 6 and ICG, and polymer albumin comprises the albumin molecule that contains sulfydryl and/or disulfide bond, between albumin molecule, by disulfide bond, interconnects, and the particle diameter of polymer albumin nanospheres is 300～400nm.

Application Example

In order to verify polymer albumin nanospheres that the embodiment of the present invention the provides effect in the medicine of preparation prevention, treatment or cancer diagnosis, this application has been studied the transmitting procedure of polymer albumin nanospheres in Mice Body and to the specificity of breast cancer tumour tissue identification and sensitivity, and the polymer albumin nanospheres that different experimental group checking embodiment is set provides suppresses the effect of tumor growth, the results are shown in Figure 6, Fig. 7 and Fig. 8.

Take MCF-7 tumor-bearing mice as model, three experimental grouies are set, do not inject polymer albumin nanospheres for first group, second group (left side tumor locus of mice) is by tail vein injection polymer albumin nanospheres aqueous solution 0.02mg/ml, but do not carry out laser irradiation, the 3rd group (the right side tumor locus of mice) is by tail vein injection polymer albumin nanospheres and carry out corresponding laser irradiation.Utilize fluorescence living imaging system (model Maestro ^tM2Maestro ^tMeX-RRO, company is U.S. CRi Maestro ^tM) and photoacoustic imaging platform (light source is tunable optical parametric oscillator, and wave-length coverage is from 400nm-2500nm, model Vibrant355II HE, Opotek, Carlsbad, USA, light source pulse repetition rate 10Hz, pulsewidth 5ns; Ultrasonic probe mid frequency 10MHz, model V315, Panametrics, Waltham, US) transmitting procedure of research polymer albumin nanospheres in Mice Body and to the specificity of breast cancer tumour tissue identification and sensitivity.

Fig. 6 and Fig. 7 are respectively polymer albumin nanospheres that embodiment 8 makes for fluorescence and the photoacoustic imaging figure of tumor bearing nude mice and tumor tissues, in Fig. 6, a does not inject polymer albumin nanospheres, in Fig. 6, b adopts the fluorescence imaging figure of the laser irradiation generation of 660nm after injection polymer albumin nanospheres 1h, c is the fluorescence imaging figure of the laser irradiation 24 rear generations of 660nm, d for using the fluorescence imaging figure producing after the laser irradiation 5min of 808nm instead on the basis of c, left side tumor locus does not carry out laser irradiation, as can be seen from Figure 6, mice left side tumor locus fluorescence signal slowly strengthens, after the tumor 660nm optical dynamic therapy of right side, fluorescence signal becomes stronger than left side, this is because adopt the laser of 660nm to irradiate right side tumor after injection 1h, Ce6 carries out optical dynamic therapy can make tumor locus produce inflammation, after irradiating 24h, polymer albumin nanospheres increases at the enriching quantity of inflammation tumor locus, adopt again the laser irradiation of 808nm, inflammation tumor is carried out to light light power to ICG and photo-thermal is treated simultaneously, after treatment 5min, right side tumor obviously weakens with respect to the fluorescence of left side tumor, the ICG that tumor locus is described substantially decomposes owing to having produced a large amount of heats in 808nm photo-thermal therapy.The acute inflammation producing after optical dynamic treatment of tumor can impel take optical agents that albumin is carrier in the further enrichment of tumor locus, has improved the targeting of polymer albumin nanospheres, has finally improved the therapeutic effect of polymer albumin nanospheres.

In Fig. 7, left figure is the photoacoustic imaging figure of the tumor tissues before the polymer albumin nanospheres that makes of injection embodiment 8, photoacoustic imaging figure when right figure is injection polymer albumin nanospheres 24h, as can be seen from the figure, left figure there is no photoacoustic signal, and right figure is stronger at tumor locus photoacoustic signal.Illustrate that polymer albumin nanospheres has very strong targeting to tumor tissues.

Fig. 8 is that the polymer albumin nanospheres that makes of embodiment 9 is for the tumor growth curve of tumor bearing nude mice light dynamic experiment.At tumor model, grow to 100mm ³after, by polymer albumin nanospheres solution by tail vein injection in Mice Body, specific experiment scheme is: by 54 nude mices (Balb/c) of 6～8 weeks, 6 one group is divided into 9 experimental grouies and tests, and comprising: 1, PBS buffer group (matched group); 2. injection only comprises the polymer albumin nanospheres (injection volume of Ce6 is 0.5mg/kg, the polymer albumin nanospheres that every 1kg injected in mice contains 0.5mg Ce6) of Ce6, after injection 1h, adopts 660nm laser irradiation 5min; The preparation that only comprises the polymer albumin nanospheres of Ce6 can be with reference to the preparation method of polymer albumin nanospheres of the present invention; The free Ce6 (injection volume of Ce6 is 0.5mg/kg, the Ce6 that every 1kg injected in mice contains 0.5mg) of 3 injection, adopts 660nm laser irradiation after injection 1h; 4. injection only comprises the polymer albumin nanospheres (injection volume of ICG is 1mg/kg, the polymer albumin nanospheres that every 1kg injected in mice contains 1mg ICG) of ICG, after injection 24h, adopts 808nm laser irradiation; The preparation that only comprises the polymer albumin nanospheres of ICG can be with reference to the preparation method of polymer albumin nanospheres of the present invention; 5. the free ICG (injection volume of ICG is 1mg/kg) of injection, adopts 808nm laser irradiation after injection 24h; 6. the free Ce6 (injection volume of Ce6 is 0.5mg/kg) of injection and free ICG (injection volume of ICG is 1mg/kg), after injection Ce6 and ICG1h, employing 660nm laser irradiation 5min, after 24h, then uses 808nm laser instead and irradiates 5min; 7. (injection volume of ICG is 1.0mg/kg to the polymer albumin nanospheres that includes Ce6 and ICG that injection embodiment 9 makes, the injection volume of Ce6 is 0.5mg/kg, the polymer albumin nanospheres that every 1kg injected in mice contains 1.0mg ICG and 0.5mg Ce6); After injection polymer albumin nanospheres 1h, adopt 660nm laser irradiation 5min, after 24h, use 808nm laser instead and irradiate 5min; 8. free Ce6 (injection volume of Ce6 is 0.25mg/kg)+ICG (injection volume of ICG is 0.5mg/kg) dissociates; After injection Ce6 and ICG1h, adopt 660nm laser irradiation 5min, after 24h, use 808nm laser instead and irradiate 5min; 9. (injection volume of Ce6 is 0.25mg/kg to the polymer albumin nanospheres that includes Ce6 and ICG that embodiment 9 makes, the injection volume of ICG is 0.5mg/kg), after injection polymer albumin nanospheres 1h, adopt 660nm laser irradiation 5min, after 24h, use 808nm laser instead and irradiate 5min.

As can be seen from Figure 8, in matched group, the volume growth speed of tumor is fast, free ICG, free Ce6, although the polymer albumin nanospheres that only comprises the polymer albumin nanospheres of ICG and only comprise Ce6 has certain inhibition to the growth of tumor, but the polymer albumin nanospheres that includes ICG and Ce6 that effect does not have the embodiment of the present invention 9 to make is effective, and along with including target in the polymer albumin nanospheres of ICG and Ce6 and deliver the increase of thing injection rate, suppress tumor effect better, with respect to matched group, experimental group 7 finally makes tumor disappear completely, can find out, the present invention first produces inflammation with the optical dynamic therapy of Ce6 at tumor locus, can impel with ICG in the further enrichment of tumor locus, adopt again ICG light power and photo-thermal to treat simultaneously, can greatly suppress the growth of tumor.

The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.

Claims

1. a polymer albumin nanospheres, is characterized in that, described polymer albumin nanospheres comprises containing the albumin molecule of sulfydryl and/or disulfide bond, between described albumin molecule, by disulfide bond, interconnect,

The particle diameter of described polymer albumin nanospheres is 10～1000nm.

2. a kind of polymer albumin nanospheres as claimed in claim 1, it is characterized in that, the described albumin molecule containing sulfydryl and/or disulfide bond is at least one in human serum albumin, bovine serum albumin, porcine hemoglobin, Recombinant Serum Albumin and hemoglobin.

3. a preparation method for polymer albumin nanospheres, is characterized in that, comprises the following steps:

The particle diameter of described polymer albumin nanospheres is 10～1000nm.

4. the preparation method of a kind of polymer albumin nanospheres as claimed in claim 3, it is characterized in that, in described step (1), the described albumin molecule containing sulfydryl and/or disulfide bond is at least one in human serum albumin, bovine serum albumin, porcine hemoglobin, Recombinant Serum Albumin and hemoglobin.

5. the preparation method of a kind of polymer albumin nanospheres as claimed in claim 3, is characterized in that, in described step (1), the quality of described target delivery thing is 0.0002～0.5 times of the described albumin quality containing sulfydryl and/or disulfide bond.

6. the preparation method of a kind of polymer albumin nanospheres as claimed in claim 3, is characterized in that, in described step (2), the described reducing agent with sulfydryl is glutathion, cysteine, mercaptoethanol or dithiothreitol, DTT.

7. the preparation method of a kind of polymer albumin nanospheres as claimed in claim 3, it is characterized in that, in described step (3), under the condition stirring, described, with the speed of 50～1000mL/s, add organic solvent to obtain microemulsion solution in carrying out ultrasonic solution.

8. the preparation method of a kind of polymer albumin nanospheres as claimed in claim 3, it is characterized in that, the described reactant liquor of step (2) shakes gently reaction at 4～60 ℃, in step (3), the reacted solution of step (2) carries out ultrasonic under the condition of 4～60 ℃, described microemulsion solution is reacted 5～240min under the condition of 4～60 ℃, and the described suspension containing polymer albumin nanospheres of step (4) is dialysed at 4～60 ℃.

9. the preparation method of a kind of polymer albumin nanospheres as claimed in claim 3, is characterized in that, in described step (3), described organic solvent is at least one in methanol, ethanol, propanol and the tert-butyl alcohol.

10. the application of polymer albumin nanospheres as claimed in claim 1 or 2 in the medicine of preparation prevention, treatment or cancer diagnosis.