CN104162171A

CN104162171A - Chlorin e6-containing polymer albumin nanosphere and preparation method and application thereof

Info

Publication number: CN104162171A
Application number: CN201410337774.3A
Authority: CN
Inventors: 蔡林涛; 盛宗海; 胡德红
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
Anticipated expiration: 2034-07-16
Also published as: CN104162165A; CN104162164B; CN104162172A; CN103495179A; CN104189916A; CN104162171B; CN104162164A; 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 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 chlorin e 6 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 to have obtained widely paying close attention to based on the drug conveying of high molecular nanometer microgranule, 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 is 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 that glutaraldehyde can 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.

Chlorin e 6 (chlorin e6, Ce6) as second filial generation photosensitizer, 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, cannot make stable water solution system, even if first use organic solvent dissolution chlorin e 6, and then be dispersed in aqueous solution, chlorin e 6 is also to exist with suspension form, is difficult to effectively be absorbed by cell; And also there is certain toxicity to cell in organic solvent itself.Therefore, how to improve the hydrophilic of Ce6, making Ce6 enter quickly and efficiently cell is the focus of studying at present to improve the curative effect of photodynamic therapy.

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, interconnects by disulfide bond, 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, interconnects by disulfide bond, 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 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 transport 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 multiple 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 ensures 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 comparatively difficulty of application, 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, there is greater advantage in biomedical applications with this nanosphere package delivering medicine, be conducive to nanosphere and enter 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 less than 60KDa mostly, polymer albumin nanospheres provided by the invention is assembled and is formed by multiple 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), the delivery efficiency that can improve the targets such as medicine and deliver things.

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 with the reducing agent of sulfydryl and obtain reactant liquor, then at 0～60 DEG C, shake is reacted 0.05～12 hour gently, 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 DEG C, described ultrasonic power bracket is 1～100KW, simultaneously under the condition stirring, add organic solvent to obtain microemulsion solution with the speed of 0.01～1000ml/s described in carrying out ultrasonic solution, described microemulsion solution is reacted after 5～240min under the condition of 0～60 DEG C, 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 DEG C 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, interconnect by disulfide bond, 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) is shake reaction gently at 4～60 DEG C, in step (3), the reacted solution of step (2) carries out ultrasonic under the condition of 4～60 DEG C, described microemulsion solution is reacted 5～240min under the condition of 4～60 DEG C, and the described suspension containing polymer albumin nanospheres of step (4) is dialysed at 4～60 DEG C.

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, the volume that the described organic solvent of step (3) 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 to be placed in pH value containing the suspension of polymer albumin nanospheres 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 the polymer albumin nanospheres of assembling is separated, 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 nanosphere 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 DEG C to pre-freeze and after 1～48 hour, is transferred at-20～-80 DEG C 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 the ethanol containing non-polar solvens such as chloroforms) 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 solvent such as size and ethanol of ultrasonic power 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.

Preferably, the preparation method of polymer albumin nanospheres provided by the invention, albumin aqueous solution and target are delivered to thing and first fully mix, make albumin carry target at bag and just deliver thing with target before delivering thing and fully contact, the efficiency that can improve albumin bag and carry target delivery thing.

The third aspect, the application of the preparation method that the invention provides the 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 provided by the invention 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, there is higher stability than the albumin carrier of physical bond;

(2) compared with the albumin carrier as stable in glutaraldehyde etc. with using chemical cross-linking agent, 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, interconnect by disulfide bond,

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

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

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);

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

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 albumin molecule, if the sulfydryl in this albumin monomer molecule or disulfide bond do not react in the time 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 60～120nm.

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

Preferably, the particle diameter of described polymer albumin nanospheres is 300～500nm.

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, described polymer albumin bag carries target and delivers the mode of thing and be: part chlorin e 6 is wrapped up by described albumin molecule, and it is intermolecular that the chlorin e 6 of remainder is embedded in described albumin.

More preferably, described chlorin e 6 is wrapped up by described albumin molecule.

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.

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

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, interconnects by disulfide bond, 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.

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 carry out real-time assessment to curative effect 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 target is delivered thing and is comprised cancer therapy drug;

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;

(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, interconnect by disulfide bond

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

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

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 to deliver the dispersibility of thing in order to increase albumin in solution, target, 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 in the time that the organic solvent and the water that adopt 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 the step that does not need stratification, 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.

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 target to deliver thing 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 of chlorin e 6 in albumin solution and/or in organic solvent.

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 (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 method of dialysing is: by step (3) gained to be placed in pH value containing the suspension of polymer albumin nanospheres be that 7～12 buffer is dialysed, obtain 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 impurity such as the inorganic molecules in the suspension of polymer albumin nanospheres on the one hand, the more important thing is, the polymer albumin nanospheres 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.

Polymer albumin nanospheres particle size range provided by the invention is between 10～1000nm, but, 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 the 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, described polymer albumin bag carries target and delivers the mode of thing and be: part chlorin e 6 is wrapped up by described albumin molecule, and the chlorin e 6 of remainder is embedded in described albumin molecule.

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 DEG C to pre-freeze and after 1～48 hour, is transferred at-20～-80 DEG C freezing 2～48 hours, then lyophilization 12～120 hours in freezer dryer.

The present invention contains the mol ratio of sulfydryl and/or disulfide bond albumin and the reducing agent with sulfydryl by control, containing the mol ratio of 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.

Preparation method of the present invention is simple, reaction condition gentleness, and reaction repeatability is good.

The 6th aspect, the application of the preparation method that the invention provides the 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, described in, be applied as the preparation method of polymer albumin nanospheres or polymer albumin nanospheres in the application of preparing in photodynamic tumor medicine, fluorescence imaging probe and photoacoustic imaging probe.

As used herein, " cancer " comprises tumor.

Brief description of the drawings

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 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 diagram of the polymer albumin nanospheres that makes of the embodiment of the present invention eight;

Fig. 8 is that the polymer albumin nanospheres that the embodiment of the present invention eight makes is injected into fluorescence and the photoacoustic imaging figure after tumor bearing nude mice;

Fig. 9 is the tumor growth curve of the polymer albumin nanospheres that makes of the embodiment of the present invention eight for tumor bearing nude mice light dynamic experiment.

Detailed description of the invention

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 DEG C, shake is reacted 1 hour gently, 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 DEG C, ultrasonic power is 100KW, simultaneously obtain microemulsion solution at the described 4mL dehydrated alcohol injecting with the speed of 1000mL/s in carrying out ultrasonic solution, described microemulsion solution is reacted after 5min under the condition of 0 DEG C, 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 DEG C, 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 DEG C to pre-freeze and after 2 hours, is transferred at negative 80 DEG C 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, interconnect by disulfide bond, 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 TEM image 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), the particle diameter of observing subsequently this polymer albumin nanospheres at different point in time sampling, 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 be in the dilution experiment approaching under physiological condition 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 the band of the albumin oligomer of this polymer albumin nanospheres of composition, band in frame 3 is albumin dimer and the trimerical band of this polymer albumin nanospheres of composition, and the band in frame 4 is the monomolecular band of albumin of this polymer albumin nanospheres of composition; 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, in the time adopting this polymer albumin nanospheres to carry out drug delivery, can be degraded by reducing substanceses such as the reductive glutathiones 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 DEG C, shake is reacted 0.05 hour gently, 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 DEG C, ultrasonic power is 1KW, simultaneously obtain microemulsion solution at the described 200mL injecting taking the speed of 0.01ml/s in carrying out ultrasonic solution containing the alcoholic solution (volume ratio of chloroform and ethanol is as 1:9) of chloroform, described microemulsion solution is reacted after 20min under the condition of 60 DEG C, leave standstill and remove organic facies after microemulsion solution layering, 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 DEG C, 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 DEG C to pre-freeze and after 1 hour, is transferred at negative 20 DEG C 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, interconnect by disulfide bond, 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, 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 DEG C, shake is reacted 12 hours gently, 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 DEG C, ultrasonic power is 100KW, simultaneously obtain microemulsion solution at the described 100mL injecting taking the speed of 1000ml/s in carrying out ultrasonic solution containing the alcoholic solution (volume ratio of chloroform and ethanol is as 1:9) of chloroform, described microemulsion solution is reacted after 240min under the condition of 30 DEG C, leave standstill and remove organic facies after microemulsion solution layering, 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 DEG C, 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 DEG C to pre-freeze and after 48 hours, is transferred at negative 50 DEG C 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, interconnect by disulfide bond, 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, then adopt the NaOH solution of 0.5mol/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 glutathion to obtain reactant liquor, then at 60 DEG C, shake is reacted 0.05 hour gently, 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 DEG C, ultrasonic power is 10KW, simultaneously obtain microemulsion solution at the described 22mL injecting taking the speed of 50ml/s in carrying out ultrasonic solution containing the alcoholic solution (volume ratio of chloroform and ethanol is as 1:9) of chloroform, described microemulsion solution is reacted after 30min under the condition of 60 DEG C, leave standstill and remove organic facies after microemulsion solution layering, 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 DEG C, 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 DEG C to pre-freeze and after 24 hours, is transferred at negative 80 DEG C 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, interconnect by disulfide bond, 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 DEG C, shake is reacted 0.05 hour gently, in described reactant liquor, the molal quantity of described cysteine is 100 times of albumin molal quantity;

(3) the reacted solution of step (2) is carried out ultrasonic under the condition of 60 DEG C, ultrasonic power is 1KW, simultaneously obtain microemulsion solution at the described 4mL injecting taking the speed of 50ml/s in carrying out ultrasonic solution containing the alcoholic solution (volume ratio of chloroform and ethanol is as 1:9) of chloroform, described microemulsion solution is reacted after 20min under the condition of 60 DEG C, leave standstill and remove organic facies after microemulsion solution layering, 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 DEG C, 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 DEG C to pre-freeze and after 1 hour, is transferred at negative 20 DEG C 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 and/or disulfide bond, between described albumin molecule, interconnects by disulfide bond.

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 DEG C, shake is reacted 12 hours gently, 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 DEG C, ultrasonic power is 100KW, simultaneously obtain microemulsion solution at the described 200mL injecting taking the speed of 1000ml/s in carrying out ultrasonic solution containing the alcoholic solution (volume ratio of chloroform and ethanol is as 1:9) of chloroform, described microemulsion solution is reacted after 240min under the condition of 30 DEG C, leave standstill and remove organic facies after microemulsion solution layering, 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 DEG C, 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 DEG C to pre-freeze and after 48 hours, is transferred at negative 80 DEG C 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, interconnect by disulfide bond, 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 DEG C, shake is reacted 17 hours gently, 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 DEG C, ultrasonic power is 10KW, simultaneously obtain microemulsion solution at the described 100mL injecting taking the speed of 0.01ml/s in carrying out ultrasonic solution containing the alcoholic solution (volume ratio of chloroform and ethanol is as 1:9) of chloroform, described microemulsion solution is reacted after 5min under the condition of 0 DEG C, leave standstill and remove organic facies after microemulsion solution layering, 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 DEG C, 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 DEG C to pre-freeze and after 24 hours, is transferred at negative 50 DEG C 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 and/or disulfide bond, between described albumin molecule, interconnects by disulfide bond.

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 DEG C, shake is reacted 0.05 hour gently, 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 DEG C, obtains the suspension containing polymer albumin nanospheres;

(4) by step (3) gained move into bag filter containing the suspension of polymer albumin nanospheres, keeping temperature is, under the condition of 25 DEG C, 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 DEG C to pre-freeze and after 2 hours, is transferred at negative 80 DEG C 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, interconnect by disulfide bond, 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 DEG C, shake is reacted 0.05 hour gently, 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 DEG C to pre-freeze and after 2 hours, is transferred at negative 80 DEG C 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, interconnect by disulfide bond, 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 DEG C, shake is reacted 2 hours gently, in described reactant liquor, the molal quantity of described glutathion is 100 times of albumin molal quantity;

(3) the reacted solution of step (2) is carried out ultrasonic under the condition of 4 DEG C, ultrasonic power is 50KW, simultaneously inject alcoholic solution with the speed of 1000mL/s described in carrying out ultrasonic solution, in this alcoholic solution, contain the chlorin e 6 that dimethyl sulfoxide and concentration are 2mg/mL, the volume ratio of the etoh solvent in volume and the alcoholic solution of dimethyl sulfoxide is 1:9, obtain microemulsion solution, this microemulsion solution obtains the suspension containing polymer albumin nanospheres react 10min under the condition of 4 DEG C 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 DEG C, 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 DEG C to pre-freeze and after 2 hours, is transferred at negative 80 DEG C 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; It is chlorin e 6 that described target is delivered thing; Polymer albumin comprises the albumin molecule that contains sulfydryl and/or disulfide bond, between albumin, interconnects by disulfide bond, and the particle diameter of polymer albumin nanospheres is 60～120nm.

The scanning electron microscope (SEM) photograph of the polymer albumin nanospheres that Fig. 7 obtains for embodiment 8, as can be seen from the figure, the particle size range of polymer albumin nanospheres is 60nm～120nm, the less and nanoparticle favorable dispersibility of particle diameter.

Embodiment nine

(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;

(2) in the albumin aqueous solution that is 12 to the described pH value of step (1) gained, add dithiothreitol, DTT to obtain reactant liquor, then at 60 DEG C, shake is reacted 240 hours gently, 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 DEG C, ultrasonic power is 1KW, simultaneously inject alcoholic solution with the speed of 1000ml/s described 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 5min under the condition of 60 DEG C, obtains the suspension containing polymer albumin nanospheres; The volume that alcoholic solution adds is 100 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 DEG C to pre-freeze and after 1 hour, is transferred at negative 20 DEG C 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; It is chlorin e 6 that described target is delivered thing; Polymer albumin comprises the albumin molecule that contains sulfydryl and/or disulfide bond, between albumin molecule, interconnects by disulfide bond, and the particle diameter of polymer albumin nanospheres is 300～400nm.

Embodiment ten

(1) get the Recombinant Serum Albumin aqueous solution that volume mass concentration is 300mg/mL (w/v), then adopt the NaOH solution of 0.1mol/L to regulate the pH value to 9 of described albumin aqueous solution;

(2) in the albumin aqueous solution that is 9 to the described pH value of step (1) gained, add beta-mercaptoethanol to obtain reactant liquor, then at 30 DEG C, shake is reacted 0.05 hour gently, 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 DEG C, ultrasonic power is 50KW, simultaneously inject alcoholic solution with the speed of 0.1ml/s described in carrying out ultrasonic solution, in alcoholic solution, contain the chlorin e 6 that dimethyl sulfoxide 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 240min under the condition of 30 DEG C, obtains the suspension of polymer albumin nanospheres; The volume that alcoholic solution adds is 10 times of the reacted liquor capacity of step (2);

(4) the polymer albumin nanospheres suspension of step (3) gained is moved into bag filter, keeping temperature is under the condition of 60 DEG C, 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 DEG C to pre-freeze and after 48 hours, is transferred at negative 50 DEG C 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; It is chlorin e 6 that described target is delivered thing; Polymer albumin comprises the albumin molecule that contains sulfydryl and/or disulfide bond, between albumin molecule, interconnects by disulfide bond, and the particle diameter of polymer albumin nanospheres is 300～500nm.

Embodiment 11

(1) get albumin mixed liquor, albumin mixed liquor contains the bovine serum albumin that concentration is 100mg/mL (w/v), chlorin e 6 and the dimethyl sulfoxide that concentration is 2mg/ml, in dimethyl sulfoxide and albumin mixed liquor, the volume ratio of aqueous solvent is 1:9, then adopts the NaOH solution of 0.1mol/L to regulate the pH value to 9 of mixed liquor;

(2) in step (1) albumin mixed liquor, add glutathion to obtain reactant liquor, then at 30 DEG C, shake is reacted 1 hour gently, and in reactant liquor, the molal quantity of glutathion is 1000 times of albumin molal quantity;

(3) the reacted solution of step (2) is carried out ultrasonic under the condition of 4 DEG C, ultrasonic power is 100KW, be injected in dehydrated alcohol carrying out the speed with 0.01mL/s in ultrasonic solution simultaneously, obtain microemulsion solution, described microemulsion solution is reacted after 10min under the condition of 4 DEG C, 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 DEG C, 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 DEG C to pre-freeze and after 2 hours, is transferred at negative 80 DEG C 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; It is chlorin e 6 that described target is delivered thing; Polymer albumin comprises the albumin molecule that contains sulfydryl and/or disulfide bond, between albumin molecule, interconnects by disulfide bond, and the particle diameter of polymer albumin nanospheres is 500～700nm.

Embodiment 12

(1) get albumin mixed liquor, in albumin mixed liquor, be the bovine serum albumin of 100mg/mL (w/v), chlorin e 6 and the dimethyl sulfoxide that concentration is 2mg/ml containing volume mass concentration, in dimethyl sulfoxide and albumin mixed liquor, the volume ratio of aqueous solvent is 1:9, then adopts the NaOH solution of 0.1mol/L to regulate the pH value to 9 of mixed liquor;

(2) in step (1) gained albumin mixed liquor, add glutathion to obtain reactant liquor, then at 30 DEG C, shake is reacted 1 hour gently, and in described reactant liquor, the molal quantity of described glutathion is 100 times of albumin molal quantity;

(3) the reacted solution of step (2) is carried out ultrasonic under the condition of 0 DEG C, ultrasonic power is 10KW, be injected in alcoholic solution carrying out the speed with 0.01mL/s in ultrasonic solution simultaneously, the chlorin e 6 that this alcoholic solution is 0.1mg/mL containing dimethyl sulfoxide and volume mass concentration, 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 10min under the condition of 0 DEG C, 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 DEG C to pre-freeze and after 2 hours, is transferred at negative 80 DEG C 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; It is chlorin e 6 that described target is delivered thing; Polymer albumin comprises the albumin molecule that contains sulfydryl and/or disulfide bond, between albumin, interconnects by disulfide bond, and the particle diameter of polymer albumin nanospheres is 700～1000nm.

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, Application Example has been studied the transmitting procedure of polymer albumin nanospheres in Mice Body and the specificity to 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 8 and Fig. 9.

Taking MCF-7 tumor-bearing mice as model, the polymer albumin nanospheres aqueous solution (concentration is 0.2mg/mL) of preparing by tail vein injection embodiment 8, then utilizes 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 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) study the transmitting procedure of polymer albumin nanospheres in Mice Body and the specificity to breast cancer tumour tissue identification and sensitivity, Fig. 8 is fluorescence and the photoacoustic imaging figure of the polymer albumin nanospheres that makes of embodiment 8 for tumor bearing nude mice tumor tissues (in dotted line frame); In Fig. 8, a and c figure are respectively the fluorescence imaging figure and the photoacoustic imaging figure that do not inject polymer albumin nanospheres, b and d figure are respectively injection fluorescence imaging figure and the photoacoustic imaging figure of polymer albumin nanospheres, as can be seen from Figure 8, while not injecting polymer albumin nanospheres, tumor locus does not all have fluorescence and photoacoustic signal, injection has fluorescence and the photoacoustic signal of tumor locus after the tumor-bearing mice 24h of polymer albumin nanospheres to be strengthened, and illustrates that polymer albumin nanospheres has very strong targeting to tumor tissues.

Fig. 9 is the tumor growth curve of the polymer albumin nanospheres that makes of embodiment 8 for tumor bearing nude mice light dynamic experiment.Specific experiment scheme: by 30 nude mices (Balb/c) of 6～8 weeks, 6 one group is divided into 5 experimental grouies and carries out the modeling of entity tumor, comprising: 1.PBS buffer group (matched group); 2. free Ce6 (injection volume of Ce6 is 2mg/kg, i.e. every 1kg injected in mice 2mg Ce6)+light group; 3. the polymer albumin nanospheres that contains Ce6 (injection volume of Ce6 is 2mg/kg, the polymer albumin nanospheres that every 1kg injected in mice contains 2mg the Ce6)+not light group that prepared by embodiment 8; 4. the polymer albumin nanospheres+light group that contains Ce6 (injection volume of Ce6 is 2mg/kg) that prepared by the embodiment 8 polymer albumin nanospheres+light group that contains Ce6 (injection volume of Ce6 is 1mg/kg) that 5. prepared by embodiment 8.Grow to 100mm at tumor model ³after, test according to the experimental program of above-mentioned 5 experimental grouies, observe tumor growth situation and draw tumor growth curve.Adopt the near-infrared laser elective irradiation mouse tumor tissue that emission wavelength is 660nm.

As can be seen from Figure 9, the volume growth speed of tumor in matched group, and the polymer albumin nanospheres that comprises Ce6 that embodiment 8 is made irradiates gross tumor volume in the experimental group of tumor tissues by tail vein injection and increases and be subject to certain inhibition in Mice Body and with near-infrared laser, and along with the concentration of Ce6 increases, tumor suppression effect is better.With respect to matched group, the gross tumor volume of experimental group 4 has reduced 70%.

To sum up, 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), simultaneously nanosphere 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, polymer albumin nanospheres also has initiatively targeting, contributes to chlorin e 6 to enter quickly and efficiently cell to improve the curative effect of photodynamic therapy.Simultaneously, 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 carry out real-time assessment to curative effect by imaging, be embodied as the photodynamic therapy of picture guiding.

The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments 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, interconnect by disulfide bond,

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

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:

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

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

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

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, add organic solvent to obtain microemulsion solution with the speed of 50～1000mL/s described 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) is shake reaction gently at 4～60 DEG C, the reacted solution of step (2) described in step (3) carries out ultrasonic under the condition of 4～60 DEG C, described microemulsion solution is reacted 5～240min under the condition of 4～60 DEG C, and the described suspension containing polymer albumin nanospheres of step (4) is dialysed at 4～60 DEG C.

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.