CN104162165A

CN104162165A - Lymphatic tracer-containing polymer albumin nanosphere and preparation method and application thereof

Info

Publication number: CN104162165A
Application number: CN201410338871.4A
Authority: CN
Inventors: 蔡林涛; 胡德红; 盛宗海
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2013-09-27
Filing date: 2014-07-16
Publication date: 2014-11-26
Also published as: CN104162164A; CN104162171A; CN104162171B; CN104189916B; CN104162172B; CN104162164B; CN104189916A; CN104162172A; CN103495179A

Abstract

The invention provides a lymphatic tracer-containing 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 is 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 lymphatic tracer 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 matter 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.

On the other hand, tumor imaging technology and the image probe of high specific, hypersensitivity are significant to cancer diagnosis.In recent years, near-infrared fluorescence imaging is developed, and at present, indocyanine-green, as near-infrared fluorescent developing agent, is applied in the clinical treatment of tumor, gastric cancer and colon cancer.What but near-infrared fluorescence imaging obtained is planar image.Image does not have the degree of depth, cannot obtain depth information, because animal viscera is deposited in together, cannot definitely judge the source of fluorescence signal.

Photoacoustic tomography is a kind of harmless 3-D formation method that development in recent years is got up, and it combines the high-contrast of pure optical imagery and the high-penetration depth characteristic of pure ultra sonic imaging, and the imaging of tissue of high-resolution and high-contrast can be provided.

Although fluorescence and photoacoustic imaging have good effect, be difficult to realize the requirement of direct observation tumor, to operation technique, bring great inconvenience.Tumor Glassless image technology, for realizing surgeon under ordinary ray, need to be by extra instrument, naked eyes are distinguished tumor tissues and normal structure, accurate tumor resection in operation, tool is of great significance.Therefore, single imaging mode is all not enough to obtain fully the information of tumor, combines and uses different kinds of molecules imaging technique can realize mutual supplement with each other's advantages each other, and comprehensively information can be provided for clarifying a diagnosis more accurately.

At present, occurred in the world the multi-modal molecular image equipment of extensive stock, yet the development of corresponding molecular image probe but lags far behind the development of image documentation equipment.The report that does not also have at present the polymer albumin nanospheres that multi-modality imaging uses.

Therefore, be necessary the polymer albumin nanospheres and the preparation method that provide a kind of multi-modality imaging to use.

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 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 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 or disulfide bond is at least one in human serum albumin, bovine serum albumin, porcine hemoglobin, Recombinant Serum Albumin and hemoglobin.

Preferably, the particle diameter of described polymer albumin nanospheres is 10～1000nm.

Preferably, the particle diameter of described polymer albumin nanospheres is 20～100nm.

Preferably, the particle diameter of described polymer albumin nanospheres is 100～200nm.

Preferably, the particle diameter of described polymer albumin nanospheres is 200～500nm.

Preferably, the particle diameter of described polymer albumin nanospheres is 600～1000nm.

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

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

As described herein, the albumin formation protein coat that in described polymer albumin nanospheres, bag year target delivery thing is preferably in nanosphere wraps up described target delivery thing.

As described herein, in described polymer albumin nanospheres, the mode of bag year target delivery thing is preferably:

Albumin in nanosphere forms protein coat, and described target is delivered thing and wrapped up by described protein coat, or

The albumin that described target is delivered in thing and nanosphere is inlayed mutually.

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 nanometer bag, carrying delivery 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 formed by a plurality of protein monomers molecular aggregatess, 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 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 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 the first organic solvent.

Further preferably, described the first organic solvent is at least one in dimethyl sulfoxide, methanol, ethanol, propanol and the tert-butyl alcohol.

Still more preferably, described the first organic solvent volume fraction in described albumin aqueous solution is 0%～20%.

Still more preferably, described the first organic solvent volume fraction in described albumin aqueous solution is 0.01%～20%.

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, the preparation method of polymer albumin nanospheres provided by the invention, albumin aqueous solution and target are delivered to first abundant mixing of thing, make albumin carry between target delivery thing and just fully contact with target delivery thing at bag, can improve albumin bag and carry the efficiency that target is delivered thing.

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.

The first organic solvent that the present invention adopts can not only 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 or the tert-butyl alcohol.

Preferably, in described step (3), the volume that described organic solvent adds is 2～100 times of the reacted solution of described step (2).

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

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, in described step (5), 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 negative 0 ℃～negative 20 ℃ to pre-freeze and after 1～48 hour, is transferred at negative 20 ℃～negative 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.

Polymer albumin nanospheres provided by the invention has under the condition of reducing agent (glutathion etc.) existence in vivo, the disulfide bond of albumin molecule is reduced into sulfydryl, the depolymerization of polymer albumin nanospheres, discharges thereby reach the object that target is delivered 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, 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 this albumin nanospheres clearly providing 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;

It is contrast agent that described target is delivered thing;

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

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 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, in described step (5), the particle diameter of described polymer albumin nanospheres is 20～100nm.

Preferably, in described step (5), the particle diameter of described polymer albumin nanospheres is 20～250nm.

Preferably, in described step (5), the particle diameter of described polymer albumin nanospheres is 20～300nm.

Preferably, in described step (5), the particle diameter of described polymer albumin nanospheres is 300～500nm.

Preferably, in described step (5), the particle diameter of described polymer albumin nanospheres is 600～1000nm.

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, described lymphatic tracer is 3, two (dimethylamino) phenothiazine-5-father-in-law chlorides (methylene blue), 6 of 7-, 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).

Preferably, to deliver the quality of thing be 0.0002～0.5 times of albumin quality in described polymer albumin nanospheres for described target.

Preferably, to deliver the quality of thing be 0.0008～0.5 times of albumin quality in described polymer albumin nanospheres for described target.

Preferably, to deliver the quality of thing be 0.008～0.5 times of albumin quality in described polymer albumin nanospheres for described target.

Preferably, described target is delivered in thing, and the mass ratio of described ICG and lymphatic tracer is 1:0.02～50.

Change the mol ratio of each component in polymer albumin nanospheres, can make the polymer albumin nanospheres that is suitable for different pharmacokineticss, those of ordinary skill can need adjustment aim to deliver the quality of thing and the mol ratio of albumin quality according to difference in the art.

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

Polymer albumin nanospheres provided by the invention is to comprise 2,7-two [1,3-dihydro-1,1-dimethyl-3-(4-sulphur butyl)-1,3,5-heptantriene list sodium salt (indocyanine-green), lymphatic tracer and albuminous complex; Polymer albumin nanospheres provided by the invention has advantages of that good stability, targeting are good, size homogeneous, good dispersion, good biocompatibility, noresidue.Polymer albumin nanospheres provided by the invention can be used for fluorescence living imaging, photoacoustic imaging and Glassless technology, realizes accurately excision in preoperative accurate location and art.In addition, polymer albumin nanospheres provided by the invention also can be used for photodynamics, the photo-thermal therapy of cancer.

As used in the present invention, " polymer albumin nanospheres " refers to comprise the multifunctional usage nano-probe that can be used for fluorescence living imaging, photoacoustic imaging and Glassless technology.

In polymer albumin nanospheres provided by the invention, described albumin can wrap and carry indocyanine-green and lymphatic tracer, and forming the stable polymer albumin shell of disulfide bond, described polymer albumin shell can improve stability, targeting and the biocompatibility of nano-probe.

Albumin molecule adopts intermolecular disulfide bond to be cross-linked to form polymer albumin shell, make nano-probe can stable existence in organism inner blood blood circulation, thereby avoid lymphatic tracer and the ICG invalid release in blood circulation transmitting procedure.

In addition, polymer albumin shell has improved the targeting of nano-probe: 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 nano-probe provides a kind of ability of passive target tumor tissues; On the other hand, tumor tissue cell's film rich surface, containing Albumin receptors such as gp60, gp30, gp18, provides a kind of ability of active target tumor tissue for having the nano-probe of albumin shell.

Secondly, the good biocompatibility of nano-probe provided by the invention, noresidue, polymer albumin shell can be in cell be degraded and discharges monomer albumin under the effect of reduced glutathion (GSH), monomer albumin is degraded by natural metabolism in vivo, thereby can not introduce in vivo any exogenous material.

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.

In polymer albumin nanospheres provided by the invention, contain the lymphatic tracers such as methylene blue, can make tumor tissues dyeing, thereby distinguish with normal structure, the targeting of the polymer albumin nanospheres of preparing by dependence the present invention is carried to tumor tissues by lymphatic tracer, and in tumor tissues enrichment, thereby can under the condition of visible ray, carry out bore hole observation, and without the instrument by extra.

In a word, it is that carrier, ICG are that optical agents, lymphatic tracer are neoplasm tracing reagent that polymer albumin nanospheres provided by the invention adopts albumin, there is good near-infrared fluorescence imaging, photoacoustic imaging and Glassless ability simultaneously, combine the advantage of fluorescence/optoacoustic and Glassless technology, contribute to realize accurately excision in preoperative accurate location and art; Polymer albumin nanospheres provided by the invention not only can be used for monitoring conveying behavior in the front nanoparticle daughter of optical therapeutic in real time, noinvasive; After optical therapeutic, also can to curative effect, carry out real-time assessment by imaging, be embodied as the optical therapeutic of picture guiding; Can also under ordinary ray, not by extra instrument, with the naked eye can distinguish tumor tissues and normal structure, and accurate tumor resection.

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

Wherein, described albumin aqueous solution is to contain the albumin mixed liquor that target is delivered thing;

It is contrast agent that described target is delivered thing;

(2) in the albumin mixed liquor 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;

(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;

It is contrast agent that described target is delivered thing;

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

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), the quality of described target delivery thing is 0.0002～0.5 times of described albumin quality.

Further preferably, to deliver the quality of thing be 0.0008～0.5 times of albumin quality in described polymer albumin nanospheres for described target.

Further preferably, in described step (1), the quality that described target is delivered thing is 0.008～0.5 times of albumin quality in described polymer albumin nanospheres.

Preferably, in described step (1), described lymphatic tracer is at least one in methylene blue, azovan blue, isosulfan blue and patent blue.

The first organic solvent that the present invention adopts not only can also improve target and deliver the dissolubility of thing (as medicine or contrast agent) in solution, and medicine or contrast agent are sufficiently uniformly dissolved.And target delivery thing is fully mixed homogeneously with protein, target is delivered thing and can fully be contacted with albumin molecule, for albumin bag in next step ultrasonic procedure carries target delivery thing, prepares.

Preferably, 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.

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 2500～5000 times of albumin molal quantity.

As used herein, described " polymer albumin " is formed by connecting by disulfide bond by monomer albumin molecule; The polymeric degree of polymerization not only with the concentration of adopted monomer albumin molecule, also relevant with the mol ratio between monomer albumin molecule with adopted reducing agent.

Preferably, the described reactant liquor of step (2) shakes gently reaction at 4～60 ℃.

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

Further preferably, institute's organic solvent also comprises at least one in dimethyl sulfoxide, chloroform, dichloromethane and normal hexane.

As described herein, in described step (3), stratification is when the organic solvent adopting and water are when immiscible, just need to carry out stratification, and get rid of organic facies; Alternately, when the organic solvent adopting and water dissolve each other, do not need stratification and remove organic facies, the organic solvent that described and water dissolves each other is preferably ethanol or DMSO.

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.

Secondly, what the present invention adopted is ultrasonic more excellent than hybrid modes such as stirrings, because the condition of ultrasonic power is more controlled.

Preferably, in described step (3), described ultrasonic power bracket is 1～100KW.

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

Preferably, in step (3), the reacted solution of step (2) carries out ultrasonic under the condition of 4～60 ℃.

Preferably, the microemulsion solution described in step (3) is reacted 5～240min under the condition of 4～60 ℃.

Preferably, in described step (3), contain target and deliver thing in described organic solvent, described target is delivered thing and is comprised 2,7-two [1,3-dihydro-1,1-dimethyl-3-(4-sulphur butyl)-1,3,5-heptantriene list sodium salt and lymphatic tracers.

Further preferably, described lymphatic tracer is at least one in methylene blue, azovan blue, isosulfan blue and patent blue.

Preferably, in described step (3), the volume that described organic solvent adds is 1～50 times of the reacted solution of described step (2).

Preferably, in described step (3), the volume that described organic solvent adds is 2～10 times of the reacted solution of described step (2).

Preferably, in described step (3), the speed of described organic solvent is incorporated as 100～1000ml/s.

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

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

Preferably, the described suspension containing polymer albumin nanospheres of step (4) is dialysed at 4～60 ℃.

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

Further preferably, in described step (4), the described pH that is for the buffer of dialysing is 7～10.

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

PBS buffer used herein or tris buffer are by conventional method configuration in industry.

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 on the one hand the impurity such as inorganic molecules in the suspension of polymer albumin nanospheres grain, the more important thing is, under alkali condition, there is exchange or reset in the disulfide bond between the polymer albumin nanospheres grain of reuniting, under alkali condition, the nanosphere that particle diameter is less be can be separated into, thereby dispersion, the uniform polymer albumin nanospheres of particle diameter obtained.

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 20～100nm, therefore, the size ratio of nano-probe prepared by the present invention is more even, and size is controlled.

Preferably, in described step (5), described polymer albumin bag carries target, and to deliver the mode of thing be that bag carries target and delivers a thing described in described polymer albumin.

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 hot air drying, parsing-desiccation, vacuum belt type drying, lyophilization, spraying is dry or distilling under reduced pressure.

Those skilled in the art can adopt the mode of different drying and dehydratings to prepare different polymer albumin nanospheres preparations as required.

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

The present invention is by controlling the mol ratio of albumin and reducing agent, albumin and target are delivered the mol ratio of thing, the pH of each step, especially the speed that the pH of dialysis buffer liquid, and organic facies and water mix obtained that particle diameter is little, particle size distribution range is narrower, controlled polymer albumin nanospheres immediately.

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, the medicine of described prevention, treatment or cancer diagnosis is the nano-probe that multi-modality imaging is used.

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, photoacoustic imaging probe or tumor Glassless image probe.

As used herein, " cancer " comprises tumor.

Polymer albumin nanospheres the invention provides and its preparation method and application has following beneficial effect: polymer albumin nanospheres provided by the invention is the multi-modal molecular image probe that integrates fluorescence imaging/photoacoustic imaging/Glassless image three functions, has overcome the drawback that single imaging is difficult to meet the comprehensive requirements such as diagnosing tumor, treatment; The preparation method of polymer albumin nanospheres provided by the invention is simple, and cost is low, can obtain that particle diameter is little, particle size distribution range is narrower, controlled polymer albumin nanospheres immediately.

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 transmission electron microscope image 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 five;

Fig. 6 is the scanning electron microscope image of the polymer albumin nanospheres that makes of the embodiment of the present invention seven;

Fig. 7 is the scanning electron microscope image of the polymer albumin nanospheres that makes of the embodiment of the present invention eight;

The polymer albumin nanospheres that Fig. 8 provides for the embodiment of the present invention eight is injected the fluorescence/visual light imaging figure before and after tumor bearing nude mice;

The polymer albumin nanospheres that Fig. 9 provides for the embodiment of the present invention eight is injected the photoacoustic imaging figure before and after tumor bearing nude mice.

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 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.

Fig. 2 is transmission electron microscope (TEM) image of the polymer albumin nanospheres that makes of the embodiment of the present invention one, and from Fig. 1 and Fig. 2, the particle diameter of polymer albumin nanospheres prepared by the embodiment of the present invention is 10～100nm.

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 315 (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 negative 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 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) dimethyl sulphoxide solution of U.S. basket and 1.5mL200 (w/v, mg/mL) Recombinant Serum Albumin is mixed, obtain albumin mixed liquor, 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 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 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 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, the 22mL simultaneously injecting in described speed of take 50ml/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 30min 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 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 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, the 4mL simultaneously injecting in described speed of take 50ml/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 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 negative 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 containing the albumin molecule of sulfydryl or disulfide bond, between described albumin molecule, by disulfide bond, interconnects.

Fig. 5 is the scanning electron microscope image of the polymer albumin nanospheres that makes of the embodiment of the present invention five, from this image, polymer albumin nanospheres size homogeneous prepared by the present embodiment, granule comparatively disperses, and 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, the 200mL 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 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 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, the 100mL 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 5min under the condition of 0 ℃, 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 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 containing the albumin molecule of sulfydryl or disulfide bond, between described albumin molecule, by disulfide bond, interconnects.

Fig. 6 is the scanning electron microscope image of the polymer albumin nanospheres that makes of the embodiment of the present invention seven, from this image, polymer albumin nanospheres size homogeneous prepared by the present embodiment, granule comparatively disperses, and 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) the 10mL ethanol solution adding in the solution of described step (2) gained obtains microemulsion solution, described microemulsion solution is reacted after 10min under the condition of 0 ℃, standingly after microemulsion solution layering, remove organic facies, obtain 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 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 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 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 bovine serum albumin solution;

(2) in the bovine serum albumin solution that is 7 to the described pH value of step (1) gained, add glutathion (2mol/L) reactant liquor, then at 0 ℃, 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 0 ℃, ultrasonic power is 80KW, (the volume mass concentration of ICG is 0.1mg/mL to the alcoholic solution containing indocyanine-green (ICG) and methylene blue simultaneously injecting in described speed of take 1000mL/s in carrying out ultrasonic solution, the volume mass concentration of methylene blue is 1mg/mL) obtain microemulsion solution, described microemulsion solution obtains the suspension containing polymer albumin nanospheres react 10min under the condition of 0 ℃ after, wherein, the volume of the described alcoholic solution adding (containing indocyanine-green (ICG) and methylene blue) is 5 times of the reacted solution 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 0 ℃, bag filter is placed in to appropriate pH dialyses 24 hours in 9 PBS buffer, within every 8 hours during this time, change liquid 1 time, each pH that adopts is 9 PBS buffer, and then bag filter is placed in appropriate 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, described polymer albumin nanospheres comprises polymer albumin and target delivery thing, described polymer albumin bag carries described target delivers thing, between described albumin molecule, by disulfide bond, interconnect, described target is delivered thing and is comprised ICG and methylene blue; The particle diameter of described polymer albumin nanospheres is 20～100nm.

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 7, Fig. 7 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.

In addition, the present embodiment also adopts fluorescence living imaging system/photoacoustic imaging platform and Glassless technology, detect transmitting procedure in polymer albumin nanospheres prepared by the present embodiment Mice Body in Mice Body and to the specificity of breast cancer tumour tissue identification and sensitivity, realize accurately excision in preoperative accurate location and art, comprise the steps:

Take MCF-7 tumor-bearing mice as model, matched group and experimental group are set, the polymer albumin nanospheres that experimental group is prepared by the tail vein injection embodiment of the present invention eight (ICG and the methylene blue of pressing 1kg mice weight injection 2mg, described ICG and methylene blue bag are loaded in polymer albumin nanospheres; Solvent is water, and concentration is 0.2mg/ml), after injection 24h, utilize fluorescence living imaging system (model Maestro ^tM2Maestro ^tMeX-RRO, company 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) and bore hole observe; Wherein, matched group is the imaging results before experimental group injection polymer albumin nanospheres.

Fluorescence/visual light imaging figure before and after the polymer albumin nanospheres injection tumor bearing nude mice that Fig. 8 provides for the embodiment of the present invention, the fluorescence imaging figure that A is matched group, the fluorescence imaging figure that B is experimental group, C be experimental group under visible ray condition, bore hole observed result.In Fig. 9, A is for adopting the photoacoustic imaging figure before polymer albumin nanospheres is injected tumor bearing nude mice, and B is the photoacoustic imaging figure of the rear 24h of injection.

As can be seen from Figure 8, the tumor locus of not injecting the nude mice of control group of polymer albumin nanospheres does not all have fluorescence signal, and the fluorescence signal of experimental group tumor locus (shown in arrow a) after injection polymer albumin nanospheres 24h is strengthened, and bore hole is visible tumor aobvious blue (shown in arrow b), tumor locus has a large amount of methylene blue enrichments, illustrates that the polymer albumin nanospheres that the embodiment of the present invention provides has very strong targeting to tumor tissues; In Fig. 9, with respect to A figure, the photoacoustic signal in B figure obviously strengthens, and in Fig. 9, A and the B position shown in a and b in corresponding diagram 8 respectively, illustrates that tumor locus has a large amount of ICG enrichments.Therefore, the polymer albumin nanospheres that the embodiment of the present invention provides can be used for fluorescence living imaging, photoacoustic imaging and Glassless technology, realizes accurately excision in preoperative accurate location and art.The polymer albumin nanospheres that the embodiment of the present invention provides can be used as tumor fluorescence/optoacoustic and Glassless affects probe, and for the diagnosis of tumor.

Embodiment nine

(1) get albumin mixed liquor, in described albumin mixed liquor, containing the volume mass concentration ICG that is 0.1mg/mL, methylene blue that volume mass concentration is 0.1mg/mL and the porcine hemoglobin of 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 (0.01mol/L) reactant liquor, then at 60 ℃, shake gently reaction 240 minutes, 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 100KW, (the volume mass concentration of ICG is 5mg/mL to the alcoholic solution containing indocyanine-green (ICG) and methylene blue simultaneously injecting in described speed of take 1000ml/s in carrying out ultrasonic solution, the volume mass concentration of methylene blue is 0.1mg/mL) obtain microemulsion solution, described microemulsion solution is reacted after 5min under the condition of 60 ℃, obtain the suspension containing polymer albumin nanospheres, wherein, the volume of the described alcoholic solution adding (containing indocyanine-green (ICG) and methylene blue) is 1 times of the reacted solution 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 appropriate pH dialyses 300 hours in 12 PBS buffer, within every 12 hours during this time, change liquid 1 time, each pH that adopts is 12 PBS buffer, and then bag filter is placed in appropriate 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 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 polymer albumin and target delivery thing, described polymer albumin bag carries described target delivers thing, between described albumin molecule, by disulfide bond, interconnect, described target is delivered thing and is comprised ICG and methylene blue; The particle diameter of described polymer albumin nanospheres is 100～200nm.

Embodiment ten

(1) get albumin mixed liquor, in described albumin mixed liquor, containing the volume mass concentration ICG that is 5mg/mL, methylene blue that volume mass concentration is 5mg/mL and the porcine hemoglobin of 100mg/mL (w/v), then adopt the NaOH solution of 0.1mol/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 (1mol/L) 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 40KW, simultaneously in the described t-butanol solution of injecting with the speed of 0.01ml/s in carrying out ultrasonic solution, obtain microemulsion solution, described microemulsion solution is reacted after 240min under the condition of 30 ℃, obtain the suspension containing polymer albumin nanospheres, the volume of the t-butanol solution adding wherein, is 100 times of the reacted solution 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 60 ℃, bag filter is placed in to appropriate pH dialyses 2 hours in 7 PBS buffer, each 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 appropriate 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 polymer albumin and target delivery thing, described polymer albumin bag carries described target delivers thing, between described albumin molecule, by disulfide bond, interconnect, described target is delivered thing and is comprised ICG and methylene blue; The particle diameter of described polymer albumin nanospheres is 200～300nm.

Embodiment 11

(1) get the porcine hemoglobin aqueous solution that volume mass concentration is 200mg/mL (w/v) (volume ratio of DMSO and water is 1:9), then adopt the NaOH solution of 10mol/L to regulate the pH value to 12 of described albumin aqueous solution;

(2) in the albumin aqueous solution that is 12 to the described pH value of step (1) gained, add dithiothreitol, DTT (0.01mol/L) reactant liquor, then at 60 ℃, shake gently reaction 240 minutes, 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 volume mass concentration of ICG is 0.02mg/mL to the alcoholic solution containing indocyanine-green (ICG) and methylene blue simultaneously injecting in described speed of take 1000ml/s in carrying out ultrasonic solution, the volume mass concentration of methylene blue is 1mg/mL) obtain microemulsion solution, described microemulsion solution is reacted after 5min under the condition of 60 ℃, obtain the suspension containing polymer albumin nanospheres, wherein, the volume of the described alcoholic solution adding (containing indocyanine-green (ICG) and methylene blue) is 10 times of the reacted solution of described step (2),

(5) the polymer albumin nanospheres solution of step (4) gained is placed at negative 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 polymer albumin and target delivery thing, described polymer albumin bag carries described target delivers thing, between described albumin molecule, by disulfide bond, interconnect, described target is delivered thing and is comprised ICG and methylene blue; The particle diameter of described polymer albumin nanospheres is 300～500nm.

Embodiment 12

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

(3) by the alcoholic solution of indocyanine-green (ICG) and methylene blue, (the volume mass concentration of ICG is 5mg/mL, the volume mass concentration of methylene blue is 0.1mg/mL) under the condition of 60 ℃, carry out ultrasonic, ultrasonic power is 1KW, simultaneously at the described reacted solution of step (2) injecting with the speed of 100ml/s in carrying out ultrasonic solution, obtain microemulsion solution, described microemulsion solution is reacted after 5min under the condition of 60 ℃, obtain the suspension containing polymer albumin nanospheres, wherein, the volume of described alcoholic solution (containing indocyanine-green (ICG) and methylene blue) is 10 times of the reacted solution of described step (2),

(5) the polymer albumin nanospheres solution of step (4) gained is placed at negative 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 polymer albumin and target delivery thing, described polymer albumin bag carries described target delivers thing, between described albumin molecule, by disulfide bond, interconnect, described target is delivered thing and is comprised ICG and methylene blue; The particle diameter of described polymer albumin nanospheres is 300～400nm.

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,

It is contrast agent that described target is delivered thing;

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 kind of polymer albumin nanospheres as claimed in claim 1, it is characterized in that, described lymphatic tracer is 3, two (dimethylamino) phenothiazine-5-father-in-law chlorides, 6 of 7-, 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, 2-((4-lignocaine) benzene) (4-(lignocaine) cyclohexane extraction-2,5-diene) methane) phenyl-Isosorbide-5-Nitrae-disulfonate, 4, two (lignocaine) triphen dehydration of 4'-methanol-2'', at least one in 4''-disulfonate sodium.

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

It is contrast agent that described target is delivered thing;

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

5. the preparation method of a kind of polymer albumin nanospheres as claimed in claim 4, 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.

6. the preparation method of a kind of polymer albumin nanospheres as claimed in claim 4, is characterized in that, in described step (1), the quality of described target delivery thing is 0.0002～0.5 times of described albumin quality.

7. the preparation method of a kind of polymer albumin nanospheres as claimed in claim 4, it is characterized in that, described lymphatic tracer is 3, two (dimethylamino) phenothiazine-5-father-in-law chlorides, 6 of 7-, 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, 2-((4-lignocaine) benzene) (4-(lignocaine) cyclohexane extraction-2,5-diene) methane) phenyl-Isosorbide-5-Nitrae-disulfonate, 4, two (lignocaine) triphen dehydration of 4'-methanol-2'', at least one in 4''-disulfonate sodium.

8. the preparation method of a kind of polymer albumin nanospheres as claimed in claim 4, is characterized in that, in described step (2), the described reducing agent with sulfydryl is glutathion, cysteine, mercaptoethanol or dithiothreitol, DTT.

9. the preparation method of a kind of polymer albumin nanospheres as claimed in claim 4, 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 preparation method of a kind of polymer albumin nanospheres as claimed in claim 4, it is characterized in that, in described step (5), the mode that the polymer albumin nanospheres solution of gained carries out drying and dehydrating processing is that lyophilization, spraying are dried or distilling under reduced pressure.

The application of the preparation method of 11. polymer albumin nanospheres as claimed in claim 1 or a kind of polymer albumin nanospheres as claimed in claim 4 in the medicine of preparation prevention, treatment or cancer diagnosis, the medicine of described prevention, treatment or cancer diagnosis is the nano-probe that multi-modality imaging is used.