CN104162171B - A kind of polymer albumin nanospheres comprising chlorin e 6 and its preparation method and application - Google Patents

A kind of polymer albumin nanospheres comprising chlorin e 6 and its preparation method and application Download PDF

Info

Publication number
CN104162171B
CN104162171B CN201410337774.3A CN201410337774A CN104162171B CN 104162171 B CN104162171 B CN 104162171B CN 201410337774 A CN201410337774 A CN 201410337774A CN 104162171 B CN104162171 B CN 104162171B
Authority
CN
China
Prior art keywords
albumin
polymer
nanospheres
solution
albumin nanospheres
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201410337774.3A
Other languages
Chinese (zh)
Other versions
CN104162171A (en
Inventor
蔡林涛
盛宗海
胡德红
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Institute of Advanced Technology of CAS
Original Assignee
Shenzhen Institute of Advanced Technology of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Institute of Advanced Technology of CAS filed Critical Shenzhen Institute of Advanced Technology of CAS
Priority to CN201410337774.3A priority Critical patent/CN104162171B/en
Publication of CN104162171A publication Critical patent/CN104162171A/en
Application granted granted Critical
Publication of CN104162171B publication Critical patent/CN104162171B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Abstract

The present invention provides a kind of polymer albumin nanospheres, the polymer albumin nanospheres include the albumin molecule containing sulfydryl and/or disulfide bond, it is connected with each other by disulfide bond between the albumin molecule, target has been contained in the polymer albumin nanospheres and has delivered thing；The target delivers thing includes cancer therapy drug；The particle diameter of the polymer albumin nanospheres is 10~1000nm.The polymer albumin nanospheres are a kind of safe and stable biocompatibility nano-carriers, can contain the targets such as medicine or contrast agent and deliver thing, and its storage stability is high, are conducive to for a long time effectively, stably discharging target delivery thing；Additionally, the particle diameter of the polymer albumin nanospheres is small, and size uniformity, good dispersion；Present invention also offers the preparation method and application of the polymer albumin nanospheres.

Description

A kind of polymer albumin nanospheres comprising chlorin e 6 and preparation method thereof and Using
The Application No. 201310449770.X of Patent Office of the People's Republic of China is submitted within 27th to this application claims in September in 2013, its hair The priority of the Chinese patent application of bright entitled " a kind of polymer albumin nanospheres and its preparation method and application ", its portion Point content is hereby incorporated by reference in the application.
Technical field
The present invention relates to biological medicine Material Field, and in particular to a kind of polymer albumin nanospheres and preparation method thereof And application.
Background technology
The rise of nanometer technology causes that the drug delivery based on high molecular nanometer particulate has obtained extensive concern, albumin With the various features such as biodegradable, nontoxic, no antigen, it is considered to be a preferable pharmaceutical carrier.And it is generally single The size of albumin molecule is not suitable for being directly used in load medicine within several nanometers, supersound method and goes the solvent method can be obtained Albumin nanospheres of the particle diameter less than 1 μm, can be used for drug delivery, but because the water solubility of albumin molecule is high, such method The Release Performance of obtained albumin nanospheres carrier is not easy to control, albumin nanoparticle is had in water good Stability, and it is difficult point in current technology of preparing not dissolved under diluting condition.
The crosslinking agents such as glutaraldehyde are often used to the nanosphere for stably obtaining, but the albumin-binding that glutaraldehyde can be non-selective The amino sites on surface, can discharge aldehydes residue in vivo, there is notable toxic and side effect to organism.Therefore, having must A kind of method for preparing safe and stable property albumin nanospheres high is provided.
Chlorin e 6 (chlorin e6, Ce6) as second generation sensitising agent, with nontoxic, tumor tissues high selectivity And nonneoplastic tissue clearance rate it is high the advantages of, singlet oxygen and/or free radical can be produced in the presence of illumination and oxygen and is used for The photodynamic therapy of tumour.But, chlorin e 6 is water insoluble, it is impossible to the water solution system of stabilization is made, even if first using Organic solvent dissolves chlorin e 6, and then in aqueous, chlorin e 6 is also exist in suspension form to redisperse, very Hardly possible is effectively absorbed by cell；And, organic solvent there is also certain toxicity to cell in itself.Therefore, how to improve Ce6's Hydrophily, makes Ce6 quickly and efficiently into cell to improve the focus that the curative effect of photodynamic therapy is current research.
The content of the invention
To solve the above problems, the invention provides a kind of polymer albumin nanospheres, the polymer albumin nano Ball includes the albumin molecule containing sulfydryl and/or disulfide bond, is connected with each other by disulfide bond between the albumin molecule, dilute There is stability higher under the conditions of releasing；Additionally, the particle diameter of the polymer albumin nanospheres is 10~1000nm, and its size is equal One, good dispersion is good carrier that the targets such as drug delivery or contrast agent deliver thing, is conducive to having for a long time in patient's body Thing is delivered in effect, safely and steadly release；Present invention also offers a kind of preparation method of polymer albumin nanospheres and should With.
In a first aspect, the invention provides a kind of polymer albumin nanospheres, the polymer albumin nanospheres bag The albumin molecule containing sulfydryl and/or disulfide bond is included, is connected with each other by disulfide bond between the albumin molecule, the poly The particle diameter of body albumin nanospheres is 10~1000nm.
Preferably, the albumin molecule containing sulfydryl and/or disulfide bond be human serum albumins, bovine serum albumin(BSA), At least one in PSA, Recombinant Serum Albumin and hemoglobin.
Preferably, target has been contained in the polymer albumin nanospheres and has delivered thing.
It is further preferred that it is at least one in cancer therapy drug and contrast agent that the target delivers thing.
It is further preferred that the cancer therapy drug is complex, 5 β of platinum and platinum, 20- epoxy -1,2 α, 4,7 β, 10 β, 13 α-[(2 ' R, 3 ' the S)-N- benzoyl -3- benzene of hexahydroxy taxane -11- alkene -9- ketone -4,10- diacetate esters -2- benzoic ethers -13 Base isoerine ester] (taxol), (7S：9S) the hydroxyls of -9- glycolyls -4- methoxyl groups -7,8,9,10- tetrahydrochysenes -6,7,9,11- four Base -7-0- (2 ', 3 ', 6 ',-three deoxidation -3 '-chloro-a-1- lysols pyranose) -5,12- naphthalenediones (adriamycin), (E, E) - 1,7- double (4- hydroxy 3-methoxybenzenes base) -1,6- heptadiene -3,5- diketone (curcumin), 1,3,5,8- tetramethyls -2,4- two (a- ethoxys) porphin phenol -6,7- dipropionic acids (haematoporphyrin), 4- ethyl -4,12- dioxy -4- hydroxyl -1H- pyrans (3', 4', 6,7) pyrrole Indoles (1,2-6) quinoline -3,14- diketone (camptothecine), (2S- is trans) -18- carboxyls -20- (carboxymethyl) -13- ethyl -2,3- Dihydro 3,7,12,17- tetramethyls -8- vinyl -21H, 23H- porphines -2- propionic acid (chlorin e 6), IR700 iodide and In chloro- carbon cyanine salt compounded of iodine (IR780) of 1,1'- diη-propyls -3,3,3', 3'- tetramethyl -10,12- trimethylenes indoles three of 11- At least one.
It is further preferred that the contrast agent be 2,7- it is double [1,3- dihydro -1,1- dimethyl -3- (4- sulphur butyls) -1,3, 5- heptantrienes mono-sodium salt (indocyanine-green), p- [(2,4- diaminourea talk endlessly pyridine -6)-N- methyl methylamino] benzoyl glutamic acid (first Aminopterin), 3,7- double (dimethylamino) phenthazine -5- father-in-law chloride (methylene blue), 6,6'- [[3,3'- dimethyl (1,1'- hexichol Base) -4,4'- diyls] double (azo groups)] double (4- amino -5- hydroxyl -1,3- naphthalenedisulfonic acids) tetrasodium salts (Evans blue), 2- ((4- lignocaines) benzene) (4- (lignocaine) hexamethylene -2,5- diene) methane) phenyl -1,4- disulfonates (isosulfan blue), In double (lignocaine) the triphen anhydrous methanols -2'', 4''- disulfonate sodium (patent blue) of 4,4'- and metal nanoparticle extremely Few one kind.
Due to albumin monomer molecule ease of solubility, the albumin nanospheres that physical method is reunited easily disintegrate, in nanosphere The medicine for containing is easy to prematurely be discharged, and is unfavorable for that the targets such as medicine deliver transport of the thing in human recycle system, i.e., The controllability of the Release Performance of the albumin nanospheres carrier that physical method is reunited is not high, and the white egg of polymer that the present invention is provided White nanosphere is connected with each other by multiple albumin monomer molecules by intermolecular disulfide bond, the polymer of this chemical bond stabilization Structure is more more stable than the protein nano ball assembled by physical method, is difficult to be diluted by human body fluid, dissolves and disintegrate, and contributes to Ensure the enough administration concentrations of targeting moiety.
In biomedical applications, the particle diameter of nano-particle medicine is important, and different particle diameter metabolic pathways also differ Sample, small particle is metabolized by kidney, and big particle diameter passes through hepatic metabolism；Wherein, the particle of 20~200nm has passive target to make tumour With the nano-particle medicine in this particle size range can reduce medicine toxic and side effect in itself, while also increasing curative effect.
Additionally, the dispersiveness of nano-particle medicine is important, poor dispersion, particle is easily reunited, or even heavy Form sediment, cause using more difficulty, particularly its biomedical applications, dispersiveness is even more important.
The present invention provide polymer albumin nanospheres particle diameter is small and size uniformity, good dispersion, with the nanosphere bag Wrap up in delivery medicine has greater advantage in biomedical applications, is conducive to nanosphere to pass through EPR effects into inside tumor and pass through Gp60 paths are targeted to tumour cell.
In addition, the molecular weight of general free protein molecule is less than 60KDa, the polymer albumin that the present invention is provided mostly Nanosphere is formed by the aggregation of multiple albumin monomer molecules, and molecular weight is higher than 60KDa, is difficult by glomerular filtration (because kidney is small Under the filtration of ball, protein molecule of the general molecular weight less than 60KDa can be disposed of during metabolism, and can not Reach target location), the delivery efficiency that the targets such as medicine deliver thing can be improved.
Second aspect, the invention provides a kind of preparation method of polymer albumin nanospheres, comprises the following steps：
(1) the albumin aqueous solution containing sulfydryl and/or disulfide bond that volume mass concentration is 0.01~300mg/mL is prepared, And it is 7~12 to adjust the pH value of the albumin aqueous solution；
(2) to adding the reducing agent with sulfydryl to obtain in the albumin aqueous solution that the pH value obtained by step (1) is 7~12 Reaction solution, is then shaken gently for reaction 0.05~12 hour at 0~60 DEG C, in the reaction solution, the going back with sulfydryl The molal quantity of former agent is 10~5000 times of albumin molar number；
(3) step (2) reacted solution is carried out into ultrasound, the power bracket of the ultrasound under conditions of 0~60 DEG C It is 1~100KW, while being added with the speed of 0.01~1000ml/s in the solution for carrying out ultrasound under conditions of stirring Organic solvent obtains microemulsion solution, after the microemulsion solution reacts 5~240min under conditions of 0~60 DEG C, stratification, so After remove organic phase, obtain the suspension containing polymer albumin nanospheres, wherein, the volume that the organic solvent is added is institute State step (2) reacted solution 1~100 times；
(4) by the suspension containing polymer albumin nanospheres obtained by step (3) 0~60 DEG C and pH value be 7~ Dialysed under conditions of 12, obtained polymer albumin nanospheres solution；
(5) the polymer albumin nanospheres solution obtained by step (4) is dried dewater treatment, obtains polymer white Protein nano ball, the polymer albumin nanospheres include the albumin molecule containing sulfydryl and/or disulfide bond, the albumin It is connected with each other by disulfide bond between molecule, the particle diameter of the polymer albumin nanospheres is 10~1000nm.
Preferably, in the step (1), the albumin molecule containing sulfydryl and/or disulfide bond be human serum albumins, At least one in bovine serum albumin(BSA), PSA, Recombinant Serum Albumin and hemoglobin.
Preferably, in the step (1), the diformazan that volume fraction is 0%~20% is contained in the albumin aqueous solution At least one in base sulfoxide, methyl alcohol, ethanol, propyl alcohol and the tert-butyl alcohol.
Dimethyl sulfoxide (DMSO), methyl alcohol, ethanol, propyl alcohol or the tert-butyl alcohol that the present invention is used can improve medicine or contrast agent in solution In solubility, be sufficiently uniformly dissolved medicine or contrast agent.
Preferably, in the step (1), thing is delivered containing target in the albumin aqueous solution, the target delivers thing Quality be 0.0002~0.5 times of the albumin quality.
It is further preferred that it is at least one in cancer therapy drug and contrast agent that the target delivers thing.
It is further preferred that the cancer therapy drug is analog, taxol, adriamycin, curcumin, the blood porphin of platinum and platinum At least one in quinoline, camptothecine, chlorin e 6, IR700 iodide and IR780.
It is further preferred that the contrast agent is indocyanine-green, methotrexate (MTX), methylene blue, Evans blue, isosulfan blue, patent At least one in blue and metal nanoparticle.
Dimethyl sulfoxide (DMSO) that the present invention is used, methyl alcohol, ethanol, propyl alcohol or the tert-butyl alcohol can also improve target and deliver thing (such as medicine Thing or contrast agent) solubility in the solution, target is delivered thing and fully dissolve and be well mixed with protein, and target delivery Thing can be fully contacted with albumin molecule, be that albumin contains target and delivers thing and prepares in next step ultrasonic procedure.
Preferably, in the step (2), the reducing agent with sulfydryl be glutathione, cysteine, mercaptoethanol or Dithiothreitol (DTT).
Preferably, in the step (3), the organic solvent is at least in methyl alcohol, ethanol, propyl alcohol and the tert-butyl alcohol Kind.
It is further preferred that also containing dimethyl sulfoxide (DMSO), chloroform, dichloromethane and n-hexane in the organic solvent In at least one.
Preferably, step (2) described reaction solution is shaken gently for reaction at 4~60 DEG C, step (2) reaction in step (3) Solution afterwards carries out ultrasound under conditions of 4~60 DEG C, and the microemulsion solution reacts 5~240min under conditions of 4~60 DEG C, Step (4) suspension containing polymer albumin nanospheres is dialysed at 4~60 DEG C.
Preferably, in the step (3), the volume that the organic solvent is added is the step (2) reacted solution 2~50 times.
It is highly preferred that the volume that adds of step (3) organic solvent for the step (2) reacted solution 2~ 20 times.
Preferably, in the step (4), the method dialysed is：First will be white containing polymer obtained by step (3) The suspension of protein nano ball is placed in the PBS that pH value is 7~12 and dialyses 10~300 hours, then in distilled water Dialysis 12 hours, obtains the solution containing polymer albumin nanospheres.
The present invention uses pH value to be dialysed for 7~12 PBS, on the one hand can remove polymer albumin nanometer The impurity such as the inorganic molecules in the suspension of rice ball, on the other hand, in the basic conditions, some are present in polymer albumin Disulfide bond between nanosphere disconnects, and then forms new disulfide bond with the protein molecule in respective nanosphere, so that The polymer albumin nanospheres of aggregation are separated, and reach the purpose of dispersion polymer albumin nanospheres, and then formed single Scattered polymer albumin nanospheres.
As described herein, the single scattered polymer albumin nanospheres are particle size range in 10~1000nm Between polymer albumin nanospheres.Under alkalescence condition before dialysis treatment, meeting between some polymer albumin nanospheres Reunite, cause the particle diameter of the nanosphere after reuniting bigger than normal, by after dialysis treatment under alkalescence condition, the nanosphere of reunion is divided Dissipate, form the smaller nanosphere of particle diameter.
The present invention uses pH value to be dialysed for 7~12 buffer solution, on the one hand can remove the suspension of nano-probe In the impurity such as inorganic molecules, it is often more important that, the nanosphere of reunion can be separated into that particle diameter is smaller to be received in the basic conditions Rice ball so that disperseed, the polymer albumin nanospheres of uniform particle sizes.
Preferably, in the step (5), target has been contained in the polymer albumin nanospheres and has delivered thing.
Preferably, the polymer albumin nanospheres solution to obtained by step (4) is dried the side of dewater treatment Formula is：Pre-freeze is shifted after 1~48 hour at polymer albumin nanospheres solution obtained by step (4) is placed in into 0~-20 DEG C To freezing 2~48 hours at -20~-80 DEG C, then freeze-drying 12~120 hours in freeze drier.
The preparation method of the polymer albumin nanospheres that the present invention is provided, employs organic solvent (second in ultrasound Alcohol or the ethanol containing non-polar solvens such as chloroforms) injection albumin solution ultrasonic emulsification-go the mode of solvent method to prepare nanometer Ball, on the one hand, protein molecule can be separated out during the organic solvent such as ethanol injection albumin solution, at the same time, albumin point Assemble to form polymer albumin nanospheres because of the formation of disulfide bond between son.On the other hand, the size of ultrasonic power is controlled With the injection rate of the organic solvent such as ethanol, the particle diameter of prepared polymer albumin nanospheres can be controlled.Because, When ultrasonic power is big, the injection rate of the organic solvent such as ethanol is big, and the particle diameter of the polymer albumin nanospheres of gained is just small；Instead It, when ultrasonic power is small, the injection rate of the organic solvent such as ethanol is small, and the particle diameter of the polymer albumin nanospheres of gained is just Greatly.Therefore, the preparation method of the polymer albumin nanospheres that the present invention is provided can not only obtain polymer albumin nanospheres, And can obtain the polymer albumin nanospheres of size tunable, the method that the present invention is provided can prepare particle diameter 10~ Polymer albumin nanospheres between 1000nm.
Preferably, the preparation method of the polymer albumin nanospheres that the present invention is provided, by the albumin aqueous solution and target Deliver thing to be first sufficiently mixed so that albumin was just delivered thing and is fully contacted before target delivery thing is contained with target, can improve Albumin contains the efficiency that target delivers thing.
The third aspect, the invention provides polymer albumin nanospheres as described in relation to the first aspect or such as second aspect institute Application of the preparation method of the polymer albumin nanospheres stated in the medicine for preparing prevention, treatment or diagnosis cancer.
As used herein, " cancer " includes tumour.
Polymer albumin nanospheres that the present invention is provided and its preparation method and application have the advantages that：
(1) the polymer albumin nanospheres that the present invention is provided by different albumin molecules by disulfide bond between molecule A nanometer pelletizing is interconnected to form, under water, phosphate buffer, ethanol, serum or culture medium equal solvent diluting condition, compares thing Managing the albumin carrier for combining has stability higher；
(2) compared with the chemical cross-linking agent such as albumin carrier of glutaraldehyde stabilization is used, the albumin that the present invention is provided Nanosphere employs protein molecule disulfide bond in itself to obtain the nanosphere of stabilization, therefore the albumin nanometer that the present invention is provided Rice ball is safer；
(3) particle diameter of the polymer albumin nanospheres that the present invention is provided is 10~1000nm, and its size uniformity, dispersion Property it is good, be good carrier that the targets such as drug delivery or contrast agent deliver thing, be conducive in patient's body for a long time effectively, peace Entirely, stably thing is delivered in release；
(4) the polymer albumin nanospheres that the present invention is provided can be used to prepare the medicine of prevention, treatment or diagnosis cancer.
Fourth aspect, the invention provides a kind of polymer albumin nanospheres, the polymer albumin nanospheres bag The albumin molecule containing sulfydryl and/or disulfide bond is included, is connected with each other by disulfide bond between the albumin molecule,
Target is contained in the polymer albumin nanospheres and has delivered thing；
The target delivers thing includes cancer therapy drug；
The cancer therapy drug includes (2S- is trans) -18- carboxyls -20- (carboxymethyl) -13- ethyl -2,3- dihydros 3,7,12, 17- tetramethyls -8- vinyl -21H, 23H- porphines -2- propionic acid (chlorin e 6)；
The particle diameter of the polymer albumin nanospheres is 10~1000nm.
As described in the present invention, " polymer albumin nanospheres " are mutually interconnected by between albumin molecule by disulfide bond Connect, due to containing at least one sulfydryl or disulfide bond in albumin molecule, if sulfydryl or two sulphur in the albumin monomer molecule Key does not react when polymer albumin nanospheres are formed, it is likely that be retained in polymer albumin ball, because This, the polymer albumin nanospheres that the present invention is provided can contain sulfydryl, disulfide bond or contain sulfydryl and disulfide bond simultaneously, The polymer albumin nanospheres that i.e. present invention is provided can contain sulfydryl and/or disulfide bond.
Preferably, the particle diameter of the polymer albumin nanospheres is 60~200nm.
Preferably, the particle diameter of the polymer albumin nanospheres is 60~120nm.
Preferably, the particle diameter of the polymer albumin nanospheres is 300~400nm.
Preferably, the particle diameter of the polymer albumin nanospheres is 300~500nm.
Preferably, the particle diameter of the polymer albumin nanospheres is 500~700nm.
Preferably, the particle diameter of the polymer albumin nanospheres is 700~1000nm.
As described herein, target delivery thing is wrapped up in the polymer albumin nanospheres and refers to the polymer albumin As carrier, contain the target and deliver thing.
Preferably, the polymer albumin contain target deliver thing mode be：Part chlorin e 6 is by described white Protein molecular is wrapped up, between the chlorin e 6 of remainder is embedded in the albumin molecule.
It is highly preferred that the chlorin e 6 is wrapped up by the albumin molecule.
Preferably, the albumin molecule containing sulfydryl and/or disulfide bond be human serum albumins, bovine serum albumin(BSA), At least one in PSA, Recombinant Serum Albumin and hemoglobin.
Preferably, the quality that the target delivers thing is the albumin quality containing sulfydryl and/or disulfide bond 0.0002~0.5 times.
Preferably, the quality that the target delivers thing is the albumin quality containing sulfydryl and/or disulfide bond 0.0008~0.5 times.
Preferably, the quality that the target delivers thing is the 0.008 of the albumin quality containing sulfydryl and/or disulfide bond ~0.5 times.
Change the mol ratio of each component in polymer albumin nanospheres, can be obtained and be suitable for many of different pharmacokinetics Aggressiveness albumin nanospheres, in the art those of ordinary skill can according to it is different need adjustment target deliver thing and albumin Mol ratio.
Albumin is a kind of biological endogenous property albumen, have the advantages that it is biodegradable, nontoxic, but albumin dilution Under the conditions of can dissolve, it is unstable in vivo, it is impossible to which that pay(useful) load drug molecule enters target organ.
Polymer albumin of the present invention includes the albumin molecule containing sulfydryl and/or disulfide bond, the albumin molecule it Between be connected with each other by disulfide bond, polymer albumin has good stability, can stable existence in aqueous, can be as good Good pharmaceutical carrier.
The present invention contains in hydrophilic polymer albumin hydrophobicity chlorin e 6, can improve chlorin The water solubility of e6 (Ce6), contributes to chlorin e 6 quickly and efficiently into cell to improve the curative effect of photodynamic therapy. Polymer albumin nanospheres of the present invention can be stable in the presence of in organism in blood circulation system, so as to avoid chlorin Invalid releases of the e6 in blood circulation system transmitting procedure；Simultaneously as the growth of tumor tissues is out of control to increased macromolecular The selectivity of class material and lipid particle, high-permeability and anelasticity (EPR effects), for polymer albumin nanospheres are provided A kind of ability of passive target tumor tissues；Tumor tissue cell's film surface is rich in the Albumin receptors such as gp60, gp30 and gp18, For polymer albumin nanospheres provide a kind of ability of active targeting tumor tissues, so as to improve polymer albumin nanometer The targeting of rice ball.
The polymer albumin nanospheres also have good near-infrared fluorescent and photoacoustic imaging ability, can be used for reality When, non-invasively nano-particle conveying behavior in vivo before monitoring treatment, curative effect can be carried out by being imaged after optical dynamic therapy Real-time assessment, is implemented as the photodynamic therapy of guiding.
Polymer albumin nanospheres particle diameter of the present invention is smaller, particle favorable dispersibility.
5th aspect, the invention provides a kind of preparation method of polymer albumin nanospheres, comprises the following steps：
(1) the albumin aqueous solution containing sulfydryl and/or disulfide bond that volume mass concentration is 0.01~300mg/mL is prepared, And it is 7~12 to adjust the pH value of the albumin aqueous solution；
Wherein, thing is delivered containing target in the albumin aqueous solution；
The target delivers thing includes cancer therapy drug；
The cancer therapy drug includes (2S- is trans) -18- carboxyls -20- (carboxymethyl) -13- ethyl -2,3- dihydros 3,7,12, 17- tetramethyl -8- vinyl -21H, 23H- porphines -2- propionic acid；
(2) to adding the reducing agent with sulfydryl to obtain in the albumin aqueous solution that the pH value obtained by step (1) is 7~12 Reaction solution, is then shaken gently for reaction 0.05~12 hour at 0~60 DEG C, in the reaction solution, the going back with sulfydryl The molal quantity of former agent is 10~5000 times of albumin molar number；
(3) step (2) reacted solution is carried out into ultrasound, the power bracket of the ultrasound under conditions of 0~60 DEG C It is 1~100KW, while being added with the speed of 0.01~1000ml/s in the solution for carrying out ultrasound under conditions of stirring Organic solvent obtains microemulsion solution, after the microemulsion solution reacts 5~240min under conditions of 0~60 DEG C, stratification, so After remove organic phase, obtain the suspension containing polymer albumin nanospheres, wherein, the volume that the organic solvent is added is institute State step (2) reacted solution 1~100 times；
(4) by the suspension containing polymer albumin nanospheres obtained by step (3) 0~60 DEG C and pH value be 7~ Dialysed under conditions of 12, obtained polymer albumin nanospheres solution；
(5) the polymer albumin nanospheres solution obtained by step (4) is dried dewater treatment, obtains polymer white Protein nano ball, the polymer albumin nanospheres include the albumin molecule containing sulfydryl and/or disulfide bond, the albumin It is connected with each other by disulfide bond between molecule,
Target is contained in the polymer albumin nanospheres and has delivered thing；
The target delivers thing includes cancer therapy drug；
The cancer therapy drug includes (2S- is trans) -18- carboxyls -20- (carboxymethyl) -13- ethyl -2,3- dihydros 3,7,12, 17- tetramethyl -8- vinyl -21H, 23H- porphines -2- propionic acid；
The particle diameter of the polymer albumin nanospheres is 10~1000nm.
Preferably, in the step (1), the albumin molecule containing sulfydryl and/or disulfide bond be human serum albumins, At least one in bovine serum albumin(BSA), PSA, Recombinant Serum Albumin and hemoglobin.
Preferably, in the step (1), the quality that the target delivers thing is described white containing sulfydryl and/or disulfide bond 0.0002~0.5 times of albumen quality.
It is further preferred that the quality that the target delivers thing is the albumin quality containing sulfydryl and/or disulfide bond 0.0008~0.5 times.
It is further preferred that in the step (1), the quality that the target delivers thing contains sulfydryl and/or two sulphur for described 0.008~0.5 times of the albumin quality of key.
Preferably, in the step (2), the molal quantity of the reducing agent with sulfydryl for albumin molar number 10~ 100 times.
Preferably, in the step (2), the molal quantity of the reducing agent with sulfydryl for albumin molar number 100~ 2500 times.
Preferably, in the step (2), the reducing agent with sulfydryl be glutathione, cysteine, mercaptoethanol or Dithiothreitol (DTT).
Preferably, in the step (2), the concentration of the reducing agent with sulfydryl is 0.01~2mol/L.
Preferably, in the step (3), the power bracket of the ultrasound is 50~100KW.
Preferably, in the step (3), the organic solvent is at least in methyl alcohol, ethanol, propyl alcohol and the tert-butyl alcohol Kind.
Alternatively, the purpose that ultrasound is carried out to solution described in step (3) of the present invention is white in solution in order to increase Albumen, target deliver the dispersiveness of thing, and are allowed to be fully contacted, and the ultrasonic step can use other hybrid modes in industry, Such as stir.
Alternatively, in the step (3), the reacted microemulsion solution can without removal organic phase the step of The suspension containing polymer albumin nanospheres can just be obtained.Stratification is when the organic solvent for using is immiscible with water When, just need to carry out stratification, and get rid of, if conversely, using the organic solvent miscible with water such as ethanol, being need not The step of stratification, removal organic phase.
It is further preferred that in the step (3), dimethyl sulfoxide (DMSO), chloroform, two are also contained in the organic solvent At least one in chloromethanes and n-hexane.
The present invention can add target to deliver thing in the albumin solution of step (1)；Alternatively, can also be in step (3) target is added to deliver thing in organic solvent；Alternatively, can simultaneously in the albumin solution of step (1), Yi Jibu Suddenly target is added to deliver thing in the organic solvent of (3).
When adding target to deliver thing in the albumin solution of step (1) and/or in step (3) organic solvent, Ke Yizai One or more in dimethyl sulfoxide (DMSO) (DMSO), tetrahydrofuran and DMF are added, to improve chlorin Solubility of the e6 in albumin solution and/or in organic solvent.
Preferably, in the step (3), the volume that the organic solvent is added is the step (2) reacted solution 2~50 times.
It is highly preferred that in the step (3), the volume that the organic solvent is added is reacted molten for the step (2) 2~20 times of liquid.
Preferably, in the step (3), the speed of the organic solvent is incorporated as 50~1000mL/s.
Organic phase (organic solvent) is added water phase (white by the preparation method of the polymer albumin nanospheres that the present invention is provided Protein solution) in, alternatively, water can be also added in organic phase.
Preferably, in the step (4), the method dialysed is：By obtained by step (3) containing the white egg of polymer The suspension of white nanosphere is placed in the buffer solution that pH value is 7~12 dialyses, and obtains polymer albumin nanospheres solution.
It is further preferred that in the step (4), the pH of the buffer solution for dialysing is 9~12.
It is further preferred that in the step (4), the buffer solution for dialysing is that PBS or Tris are buffered Liquid.
It is further preferred that in the step (4), the suspension containing polymer albumin nanospheres is in buffer solution Dialysis time be 10~300 hours.
It is further preferred that in the step (4), the suspension containing polymer albumin nanospheres be placed in pH value for 7~ After being dialysed in 12 buffer solution, then it is placed in dialysis in distilled water.
It is further preferred that the time for being placed in dialysis in distilled water is 1~24 hour.
The present invention uses pH value to be dialysed for 7~12 PBS, on the one hand can remove albumin containing polymer The impurity such as the inorganic molecules in the suspension of nanosphere, it is often more important that, the polymer albumin nanospheres of reunion are in alkalescence Under the conditions of can be separated into the smaller nanosphere of particle diameter so that disperseed, the polymer albumin nanospheres of uniform particle sizes.
Preferably, step (2) described reaction solution is shaken gently for reaction at 4~60 DEG C, step (2) reaction in step (3) Solution afterwards carries out ultrasound under conditions of 4~60 DEG C, and the microemulsion solution reacts 5~240min under conditions of 4~60 DEG C, Step (4) suspension containing polymer albumin nanospheres is dialysed at 4~60 DEG C.
The present invention provide polymer albumin nanospheres particle size range between 10~1000nm, however, this is not same The particle diameter distribution of nano-probe prepared by one batch, conversely, each batch obtained using the preparation method that the present invention is provided The particle size distribution range of nano-probe is narrower, such as between 20~250nm, therefore, the grain of nano-probe prepared by the present invention Footpath is than more uniform, and size is controllable.
Preferably, the polymer albumin contain target deliver thing mode be：Part chlorin e 6 is by described white Protein molecular is wrapped up, and the chlorin e 6 of remainder is embedded in the albumin molecule.
It is highly preferred that the chlorin e 6 is wrapped up by the albumin molecule.
Preferably, in the step (5), the polymer albumin nanospheres solution to obtained by step (4) is done The mode of dry dewater treatment is freeze-drying, spray drying or vacuum distillation.
It is it is further preferred that in the step (5), the step of the freeze-drying：By the polymer obtained by step (4) Albumin nanospheres solution be placed in 0~-20 DEG C at pre-freeze be transferred to -20~-80 DEG C after 1~48 hour at freeze 2~48 hours, Then freeze-drying 12~120 hours in freeze drier.
Present invention mol ratio containing sulfydryl and/or disulfide bond albumin and the reducing agent with sulfydryl by control, containing sulfydryl And/or disulfide bond albumin and target deliver the mol ratio of thing, the pH of the pH of each step, especially elution buffer, Yi Jiyou The speed that machine is mutually mixed with water conjunction obtains that particle diameter is small, particle size distribution range is narrower, size tunable polymer albumin nano Ball.
Preparation method of the present invention is simple, and reaction condition is gentle, and reaction reappearance is good.
6th aspect, the invention provides the polymer albumin nanospheres as described in fourth aspect or such as institute in terms of the 5th Application of the preparation method of the polymer albumin nanospheres stated in the medicine for preparing prevention, treatment or diagnosis cancer.
Preferably, the application is the preparation method of polymer albumin nanospheres or polymer albumin nanospheres in system Application in standby photodynamic therapy medicine, fluorescence imaging probe and photoacoustic imaging probe.
As used herein, " cancer " includes tumour.
Brief description of the drawings
Fig. 1 is the scanning electron microscope image of polymer albumin nanospheres obtained in the embodiment of the present invention one；
Fig. 2 is the images of transmissive electron microscope of polymer albumin nanospheres obtained in the embodiment of the present invention one；
Fig. 3 is dilution experiment of the polymer albumin nanospheres in different solvents obtained in the embodiment of the present invention one；
Fig. 4 is polymer albumin nanospheres dissolution experiment under the conditions of reduced form obtained in the embodiment of the present invention one；
Fig. 5 is the scanning electron microscope image of polymer albumin nanospheres obtained in the embodiment of the present invention five；
Fig. 6 is the scanning electron microscope image of polymer albumin nanospheres obtained in the embodiment of the present invention seven；
Fig. 7 is the scanning electron microscope diagram of polymer albumin nanospheres obtained in the embodiment of the present invention eight；
Fig. 8 is injected into fluorescence and light after tumor bearing nude mice for polymer albumin nanospheres obtained in the embodiment of the present invention eight Acoustic imaging figure；
Fig. 9 is that polymer albumin nanospheres obtained in the embodiment of the present invention eight are used for the swollen of tumor bearing nude mice light dynamic experiment Knurl growth curve.
Specific embodiment
As described below is the preferred embodiment of the present invention, it is noted that for those skilled in the art For, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications are also considered as Protection scope of the present invention.
Embodiment one
A kind of preparation method of polymer albumin nanospheres, comprises the following steps：
(1) dimethyl sulphoxide solution and 1mL of the indocyanine-green that 1mL volume mass concentration is 0.01 (w/v, mg/mL) are taken Volume mass concentration mixes for the bovine serum albumin(BSA) of 0.02 (w/v, mg/mL), obtains albumin mixed liquor, then uses The NaOH solution of 0.1mol/L adjusts the pH value of the albumin mixed liquor to 7；
(2) to adding glutathione to obtain reaction solution in the albumin mixed liquor that the pH value obtained by step (1) is 7, so Reaction 1 hour is shaken gently at 0 DEG C afterwards, in the reaction solution, the molal quantity of the glutathione is albumin molar number 10 times；
(3) step (2) reacted solution is carried out into ultrasound under conditions of 0 DEG C, ultrasonic power is 100KW, while The 4mL absolute ethyl alcohols injected with the speed of 1000mL/s in the solution for carrying out ultrasound obtain microemulsion solution, and the micro emulsion is molten After liquid reacts 5min under conditions of 0 DEG C, the suspension containing polymer albumin nanospheres is obtained；
(4) suspension containing polymer albumin nanospheres obtained by step (3) is moved into bag filter, keeping temperature is 0 Under conditions of DEG C, bag filter is placed in the PBS that 5L pH are 10 and is dialysed 10 hours, period every 12 hours change liquid 1 Secondary, it is 10 PBS that 5L pH are used every time, bag filter is placed in 5L distilled waters again then and is dialysed 1 hour, is obtained Polymer albumin nanospheres solution；
(5) pre-freeze is transferred to after 2 hours at the polymer albumin nanospheres solution obtained by step (4) being placed in into minus 20 DEG C Freezed 12 hours at minus 80 DEG C, then freeze-drying 12 hours in freeze drier, obtain polymer albumin nanospheres, institute Stating polymer albumin nanospheres includes the albumin molecule containing sulfydryl and/or disulfide bond, passes through between the albumin molecule Disulfide bond is connected with each other, and the particle diameter of the polymer albumin nanospheres is 10~100nm.
To absolutely prove the beneficial effect of polymer albumin nanospheres prepared by the embodiment of the present invention, the present embodiment is also carried The scanning electron microscope image of the polymer albumin nanospheres is supplied, as shown in figure 1, Fig. 1 makes for the embodiment of the present invention one The scanning electron microscope image of the polymer albumin nanospheres for obtaining, from the image, polymer manufactured in the present embodiment Albumin nanospheres size uniformity, particle more disperses.
The present embodiment additionally provides transmission electron microscope (TEM) image of the polymer albumin nanospheres, as shown in Fig. 2 Fig. 2 The TEM image of polymer albumin nanospheres obtained in the embodiment of the present invention one, it is manufactured in the present embodiment from the image The particle diameter of polymer albumin nanospheres is 10~100nm, and size uniformity, particle more disperses.
Additionally, the present embodiment additionally provides the dilution experiment of the polymer albumin nanospheres in different solvents, specifically Operate and be：The polymer albumin nanospheres solution is dissolved in into phosphate buffer (pH is 7.4), serum respectively, and (pH is 7.4) and in cell culture medium (pH is 7.4), the polymer albumin nanospheres then are observed in different point in time sampling Particle diameter, as a result as shown in Figure 3.
Fig. 3 is dilution experiment of the polymer albumin nanospheres in different solvents obtained in the embodiment of the present invention one, by Fig. 3 understands：The particle diameter of the polymer albumin nanospheres in phosphate buffer, serum and cell culture medium is changed over time Not substantially, i.e., the polymer albumin nanospheres that the present invention is provided can stably be deposited in the dilution experiment under close to physiological condition With medical application prospect.
Secondly, the present embodiment additionally provides the dissolution experiment of the polymer albumin nanospheres under the conditions of reduced form, tool Gymnastics conduct：The polymer albumin nanospheres solution is dissolved in the dithiothreitol (DTT) that concentration is 20mM respectively, 2 hours, 8 is small When and after 24 hours, respectively run SDS-PAGE electrophoresis, detect the dissolving of the polymer albumin nanospheres under the conditions of reduced form Rule, wherein, a, b, c swimming lane correspond to sample of the polymer albumin nanospheres behind reductase 12 hour, 8 hours, 24 hours respectively Product, d swimming lanes are the control of albumin monomer molecule, as a result as shown in Figure 4.
Fig. 4 is polymer albumin nanospheres dissolution experiment under the conditions of reduced form obtained in the embodiment of the present invention one, by Fig. 4 understands that the band in frame 1 is polymer albumin nanospheres manufactured in the present embodiment, and the band in frame 2 is the composition poly The band of the albumin oligomer of body albumin nanospheres, the band in frame 3 be constitute the polymer albumin nanospheres it is white The band of albumen dimer and tripolymer, the band in frame 4 is the albumin unimolecule for constituting the polymer albumin nanospheres Band；The result shows, i.e., the band concentration in the corresponding frame 3 of albumin dimer and tripolymer with the recovery time extension And raise, additionally, the band concentration in the corresponding frame 4 of protein monomers is also significantly improved with the extension of recovery time；I.e. The polymer albumin nanospheres that the present embodiment is provided can dissolve under conditions of reducing agent presence, and with the recovery time Extension, the degree that its disulfide bond is reduced is higher, therefore, when drug delivery is carried out using the polymer albumin nanospheres, Can be degraded by reducing substanceses such as the reductive glutathiones in cell, so as to discharge medicine.
Embodiment two
A kind of preparation method of polymer albumin nanospheres, comprises the following steps：
(1) dimethyl sulphoxide solution and 2mL volumes that 0.1mL volume mass concentration is 0.1 (w/v, mg/mL) adriamycin are taken Mass concentration mixes for the PSA of 300 (w/v, mg/mL), albumin mixed liquor is obtained, then using 1mol/L's NaOH solution adjusts the pH value of the albumin mixed liquor to 7；
(2) reaction solution is obtained to addition dithiothreitol (DTT) in the albumin mixed liquor that the pH value obtained by step (1) is 7, Then reaction 0.05 hour is shaken gently at 60 DEG C, in the reaction solution, the molal quantity of the dithiothreitol (DTT) is white egg 5000 times of white molal quantity；
(3) step (2) reacted solution is carried out into ultrasound under conditions of 60 DEG C, ultrasonic power is 1KW, while in institute State the ethanol solution (bodies of chloroform and ethanol of the 200mL containing chloroform injected with the speed of 0.01ml/s in the solution for carrying out ultrasound Product is than being 1:9) microemulsion solution is obtained, after the microemulsion solution reacts 20min under conditions of 60 DEG C, standing treats microemulsion solution point Organic phase is removed after layer, the suspension containing polymer albumin nanospheres is obtained；
(4) suspension containing polymer albumin nanospheres obtained by step (3) is moved into bag filter, keeping temperature is 30 Under conditions of DEG C, bag filter is placed in the PBS that 1L pH are 7 and is dialysed 300 hours, period every 12 hours change liquid 1 Secondary, it is 7 PBS that 1L pH are used every time, bag filter is placed in 5L distilled waters again then and is dialysed 24 hours, is obtained Polymer albumin nanospheres solution；
(5) pre-freeze is transferred to negative at the polymer albumin nanospheres solution obtained by step (4) being placed in into 0 DEG C after 1 hour Freezed 2 hours at 20 DEG C, then freeze-drying 72 hours in freeze drier, obtain polymer albumin nanospheres, described Polymer albumin nanospheres include the albumin molecule containing sulfydryl and/or disulfide bond, and two are passed through between the albumin molecule Sulfide linkage is connected with each other, and the particle diameter of the polymer albumin nanospheres is 900~1000nm.
Embodiment three
A kind of preparation method of polymer albumin nanospheres, comprises the following steps：
(1) it is the aqueous solution and 1.5mL200 (w/v, mg/ of 0.5 (w/v, mg/mL) U.S.s basket to take 0.5mL volume mass concentration ML Recombinant Serum Albumin mixing), obtains albumin mixed liquor, then adjusts the white egg using the NaOH solution of 2mol/L The pH value of white mixed liquor is to 12；
(2) reaction solution is obtained to addition beta -mercaptoethanol in the albumin mixed liquor that the pH value obtained by step (1) is 12, Then reaction 12 hours is shaken gently at 30 DEG C, in the reaction solution, the molal quantity of the beta -mercaptoethanol is albumin 100 times of molal quantity；
(3) step (2) reacted solution is carried out into ultrasound under conditions of 30 DEG C, ultrasonic power is 100KW, while Ethanol solutions of the 100mL injected with the speed of 1000ml/s in the solution for carrying out ultrasound containing chloroform be (chloroform and ethanol Volume ratio is 1:9) microemulsion solution is obtained, after the microemulsion solution reacts 240min under conditions of 30 DEG C, standing treats that micro emulsion is molten Organic phase is removed after liquid layering, the suspension containing polymer albumin nanospheres is obtained；
(4) suspension containing polymer albumin nanospheres obtained by step (3) is moved into bag filter, keeping temperature is 60 Under conditions of DEG C, bag filter is placed in the PBS that 1L pH are 12 and is dialysed 144 hours, it is 12 that 1L pH are used every time PBS, period every 12 hours change liquid 1 time, bag filter is placed in 5L distilled waters again then and is dialysed 12 hours, obtain Polymer albumin nanospheres solution,
(5) pre-freeze is transferred to after 48 hours at the polymer albumin nanospheres solution obtained by step (4) being placed in into minus 4 DEG C Freezed 48 hours at minus 50 DEG C, then freeze-drying 96 hours in freeze drier, obtain polymer albumin nanospheres, institute Stating polymer albumin nanospheres includes the albumin molecule containing sulfydryl and/or disulfide bond, passes through between the albumin molecule Disulfide bond is connected with each other, and the particle diameter of the polymer albumin nanospheres is 100~200nm.
Example IV
A kind of preparation method of polymer albumin nanospheres, comprises the following steps：
(1) take 1mL volume mass concentration be 1 (w/v, mg/mL) chlorin e 6 solution dimethyl sulphoxide solution and The hemoglobin solutions mixing of 1mL300 (w/v, mg/mL), obtains albumin mixed liquor, and then the NaOH using 0.5mol/L is molten Liquid adjusts the pH value of the albumin mixed liquor to 9；
(2) to adding glutathione to obtain reaction solution in the albumin mixed liquor that the pH value obtained by step (1) is 9, so Reaction 0.05 hour is shaken gently at 60 DEG C afterwards, and in the reaction solution, the molal quantity of the glutathione rubs for albumin 2500 times of that number；
(3) step (2) reacted solution is carried out into ultrasound under conditions of 60 DEG C, ultrasonic power is 10KW, while Ethanol solution (the volumes of chloroform and ethanol of the 22mL containing chloroform injected with the speed of 50ml/s in the solution for carrying out ultrasound Than being 1:9) microemulsion solution is obtained, after the microemulsion solution reacts 30min under conditions of 60 DEG C, standing treats that microemulsion solution is layered After remove organic phase, obtain the suspension containing polymer albumin nanospheres；
(4) suspension containing polymer albumin nanospheres obtained by step (3) is moved into bag filter, keeping temperature is 60 Under conditions of DEG C, bag filter is placed in the PBS that 1L pH are 9 and is dialysed 36 hours, period every 12 hours change liquid 1 time, It is 9 PBS to use 1L pH every time, bag filter is placed in 5L distilled waters again then and is dialysed 18 hours, obtains poly Body albumin nanospheres solution；
(5) pre-freeze is shifted after 24 hours at the polymer albumin nanospheres solution obtained by step (4) being placed in into minus 10 DEG C Freezed 24 hours to minus 80 DEG C, then freeze-drying 120 hours in freeze drier, obtain polymer albumin nano Ball, the polymer albumin nanospheres include the albumin molecule containing sulfydryl and/or disulfide bond, between the albumin molecule It is connected with each other by disulfide bond, the particle diameter of the polymer albumin nanospheres is 600~700nm.
Embodiment five
A kind of preparation method of polymer albumin nanospheres, comprises the following steps：
(1) the PSA solution that 2mL volume mass concentration is 0.01 (w/v, mg/mL) is taken, then using 1mol/ The NaOH solution of L adjusts the pH value of the albumin mixed liquor to 7；
(2) to adding cysteine to obtain reaction solution in the albumin mixed liquor that the pH value obtained by step (1) is 7, so Reaction 0.05 hour is shaken gently at 60 DEG C afterwards, and in the reaction solution, the molal quantity of the cysteine rubs for albumin 100 times of that number；
(3) step (2) reacted solution is carried out into ultrasound under conditions of 60 DEG C, ultrasonic power is 1KW, while in institute State the ethanol solution (volume ratios of chloroform and ethanol of the 4mL containing chloroform injected with the speed of 50ml/s in the solution for carrying out ultrasound It is 1:9) microemulsion solution is obtained, after the microemulsion solution reacts 20min under conditions of 60 DEG C, is stood after after microemulsion solution layering Organic phase is removed, the suspension containing polymer albumin nanospheres is obtained；
(4) suspension containing polymer albumin nanospheres obtained by step (3) is moved into bag filter, keeping temperature is 30 Under conditions of DEG C, bag filter is placed in the PBS that 1L pH are 7 and is dialysed 10 hours, period every 12 hours change liquid 1 time, It is 7 PBS to use 1L pH every time, bag filter is placed in 5L distilled waters again then and is dialysed 1 hour, obtains poly Body albumin nanospheres solution；
(5) pre-freeze is transferred to negative at the polymer albumin nanospheres solution obtained by step (4) being placed in into 0 DEG C after 1 hour Freezed 2 hours at 20 DEG C, then freeze-drying 12 hours in freeze drier, obtain polymer albumin nanospheres, described Polymer albumin nanospheres include the albumin molecule containing sulfydryl and/or disulfide bond, and two are passed through between the albumin molecule Sulfide linkage is connected with each other.
Fig. 5 is the scanning electron microscope image of polymer albumin nanospheres obtained in the embodiment of the present invention five, by this Image understands that polymer albumin nanospheres size uniformity manufactured in the present embodiment, particle more disperses, the white egg of polymer The particle diameter of white nanosphere is 700~800nm.
Embodiment six
A kind of preparation method of polymer albumin nanospheres, comprises the following steps：
(1) the Recombinant Serum Albumin solution of 2mL300 (w/v, mg/mL) is taken, is then adjusted using the NaOH solution of 2mol/L Save the pH value of the albumin mixed liquor to 12；
(2) reaction solution is obtained to addition dithiothreitol (DTT) in the albumin mixed liquor that the pH value obtained by step (1) is 12, Then reaction 12 hours is shaken gently at 30 DEG C, in the reaction solution, the molal quantity of the dithiothreitol (DTT) is albumin 5000 times of molal quantity；
(3) step (2) reacted solution is carried out into ultrasound under conditions of 30 DEG C, ultrasonic power is 100KW, while Ethanol solutions of the 200mL injected with the speed of 1000ml/s in the solution for carrying out ultrasound containing chloroform be (chloroform and ethanol Volume ratio is 1:9) microemulsion solution is obtained, after the microemulsion solution reacts 240min under conditions of 30 DEG C, standing treats that micro emulsion is molten Organic phase is removed after liquid layering, the suspension containing polymer albumin nanospheres is obtained；
(4) suspension containing polymer albumin nanospheres obtained by step (3) is moved into bag filter, keeping temperature is 60 Under conditions of DEG C, bag filter is placed in the PBS that 1L pH are 12 and is dialysed 300 hours, it is 12 that 1L pH are used every time PBS, period every 12 hours change liquid 1 time, bag filter is placed in 5L distilled waters again then and is dialysed 24 hours, obtain Polymer albumin nanospheres solution；
(5) pre-freeze is transferred to after 48 hours at the polymer albumin nanospheres solution obtained by step (4) being placed in into minus 4 DEG C Freezed 36 hours at minus 80 DEG C, then freeze-drying 96 hours in freeze drier, obtain polymer albumin nanospheres, institute Stating polymer albumin nanospheres includes the albumin molecule containing sulfydryl and/or disulfide bond, passes through between the albumin molecule Disulfide bond is connected with each other, and the particle diameter of the polymer albumin nanospheres is 10~100nm.
Embodiment seven
A kind of preparation method of polymer albumin nanospheres, comprises the following steps：
(1) albumin solution of 2mL150 (w/v, mg/mL) is taken, then the NaOH solution regulation using 2mol/L is described white The pH value of mixed liquid of protein is to 9；
(2) reaction solution is obtained to addition beta -mercaptoethanol in the albumin mixed liquor that the pH value obtained by step (1) is 9, Then reaction 17 hours is shaken gently at 0 DEG C, in the reaction solution, the molal quantity of the beta -mercaptoethanol rubs for albumin 2500 times of that number；
(3) step (2) reacted solution is carried out into ultrasound under conditions of 0 DEG C, ultrasonic power is 10KW, while in institute State the ethanol solution (bodies of chloroform and ethanol of the 100mL containing chloroform injected with the speed of 0.01ml/s in the solution for carrying out ultrasound Product is than being 1:9) microemulsion solution is obtained, after the microemulsion solution reacts 5min under conditions of 0 DEG C, standing treats that microemulsion solution is layered After remove organic phase, obtain the suspension containing polymer albumin nanospheres；
(4) suspension containing polymer albumin nanospheres obtained by step (3) is moved into bag filter, keeping temperature is 0 Under conditions of DEG C, bag filter is placed in the PBS that 1L pH are 9 and is dialysed 144 hours, it is 12 that 1L pH are used every time PBS, period every 12 hours change liquid 1 time, bag filter is placed in 5L distilled waters again then and is dialysed 12 hours, obtain many Aggressiveness albumin nanospheres solution；
(5) pre-freeze is shifted after 24 hours at the polymer albumin nanospheres solution obtained by step (4) being placed in into minus 20 DEG C Freezed 48 hours to minus 50 DEG C, then freeze-drying 120 hours in freeze drier, obtain polymer albumin nano Ball, the polymer albumin nanospheres include the albumin molecule containing sulfydryl and/or disulfide bond, between the albumin molecule It is connected with each other by disulfide bond.
Fig. 6 is the scanning electron microscope image of polymer albumin nanospheres obtained in the embodiment of the present invention seven, by this Image understands that polymer albumin nanospheres size uniformity manufactured in the present embodiment, particle more disperses, the white egg of polymer The particle diameter of white nanosphere is 700~900nm.
Comparative example one
A kind of preparation method of polymer albumin nanospheres, comprises the following steps：
(1) bovine serum albumin solution of 2mL0.01 (w/v, mg/mL) is taken；
(2) to adding cysteine to obtain reaction solution in the Bovine Serum Albumin in Aqueous Solution obtained by step (1), then at 60 DEG C Under be shaken gently for reaction 0.05 hour, in the reaction solution, the molal quantity of the cysteine is the 10 of albumin molar number Times；
(3) 10mL ethanol solutions are added to obtain microemulsion solution, the micro emulsion in the solution obtained by the step (2) After solution reacts 10min under conditions of 0 DEG C, the suspension containing polymer albumin nanospheres is obtained；
(4) suspension containing polymer albumin nanospheres obtained by step (3) is moved into bag filter, keeping temperature is 25 Under conditions of DEG C, bag filter is placed in 5L distilled waters and is dialysed 12 hours, obtain polymer albumin nanospheres solution；
(5) pre-freeze is transferred to after 2 hours at the polymer albumin nanospheres solution obtained by step (4) being placed in into minus 20 DEG C Freezed 24 hours at minus 80 DEG C, then freeze-drying 48 hours in freeze drier, obtain polymer albumin nanospheres, institute Stating polymer albumin nanospheres includes the albumin molecule containing sulfydryl and/or disulfide bond, passes through between the albumin molecule Disulfide bond is connected with each other, and the particle diameter of the polymer albumin nanospheres is 10~600nm.
Comparative example two
A kind of preparation method of polymer albumin nanospheres, comprises the following steps：
(1) it is the indocyanine-green of 1 (w/v, mg/mL) and the ox blood of 1mL300 (w/v, mg/mL) to take 1mL volume mass concentration Pure albumen mixing, obtains albumin mixed liquor；
(2) to adding glutathione to obtain reaction solution in the albumin mixed liquor obtained by step (1), then at 60 DEG C gently Shake reaction 0.05 hour, in the reaction solution, the molal quantity of the glutathione is 5000 times of albumin molar number；
(3) 10mL ethanol solutions are added to obtain microemulsion solution, the micro emulsion in the solution obtained by the step (2) After solution reacts 10min under conditions of 0 DEG C, the suspension containing polymer albumin nanospheres is obtained；
(4) suspension containing polymer albumin nanospheres obtained by step (3) is moved into bag filter, keeping temperature is 25 Under conditions of DEG C, bag filter is placed in 5L distilled waters and is dialysed 12 hours, obtain polymer albumin nanospheres solution；
(5) pre-freeze is transferred to after 2 hours at the polymer albumin nanospheres solution obtained by step (4) being placed in into minus 20 DEG C Freezed 24 hours at minus 80 DEG C, then freeze-drying 48 hours in freeze drier, obtain polymer albumin nanospheres, institute Stating polymer albumin nanospheres includes the albumin molecule containing sulfydryl and/or disulfide bond, passes through between the albumin molecule Disulfide bond is connected with each other, and the particle diameter of the polymer albumin nanospheres is 100~1000nm.
Relative to polymer albumin nanospheres provided in an embodiment of the present invention, comparative example 1 and comparative example 2 carry The polymer albumin nanospheres centralized particle diameter degree of confession is not high, is unfavorable for its application in biomedicine.
Embodiment eight
A kind of preparation method of polymer albumin nanospheres, comprises the following steps：
(1) Bovine Serum Albumin in Aqueous Solution that volume mass concentration is 50mg/mL (w/v) is taken, then using 0.1mol/L NaOH solution adjust the pH value of the albumin aqueous solution to 7；
(2) to adding glutathione to obtain reaction solution in the albumin aqueous solution that the pH value obtained by step (1) is 7, so Reaction 2 hours is shaken gently at 4 DEG C afterwards, in the reaction solution, the molal quantity of the glutathione is albumin molar number 100 times；
(3) step (2) reacted solution is carried out into ultrasound under conditions of 4 DEG C, ultrasonic power is 50KW, while in institute Stating is carried out injecting ethanol solution with the speed of 1000mL/s in ultrasonic solution, and dimethyl sulfoxide (DMSO) and concentration are contained in the ethanol solution It is the chlorin e 6 of 2mg/mL, the volume ratio of the etoh solvent in the volume and ethanol solution of dimethyl sulfoxide (DMSO) is 1：9, obtain Microemulsion solution, the microemulsion solution obtains the suspension containing polymer albumin nanospheres after reacting 10min under conditions of 4 DEG C； The volume that ethanol solution is added is 2 times of the step (2) reacted liquor capacity；
(4) suspension containing polymer albumin nanospheres obtained by step (3) is moved into bag filter, keeping temperature is 4 Under conditions of DEG C, bag filter is placed in the PBS that 1L pH are 9 and is dialysed 24 hours, period every 8 hours change liquid 1 time, It is 9 PBS to use 1L pH every time, bag filter is placed in 1L distilled waters again then and is dialysed 12 hours, obtains poly Body albumin nanospheres solution；
(5) pre-freeze is transferred to after 2 hours at the polymer albumin nanospheres solution obtained by step (4) being placed in into minus 20 DEG C Freezed 24 hours at minus 80 DEG C, then freeze-drying 48 hours in freeze drier, obtain polymer albumin nanospheres, institute State and contained in polymer albumin nanospheres target and deliver thing；It is chlorin e 6 that the target delivers thing；The white egg of polymer The white albumin molecule included containing sulfydryl and/or disulfide bond, is connected with each other, polymer albumin between albumin by disulfide bond The particle diameter of nanosphere is 60~120nm.
Fig. 7 is the scanning electron microscope (SEM) photograph of the polymer albumin nanospheres obtained by embodiment 8, it can be seen that many The particle size range of aggressiveness albumin nanospheres is 60nm~120nm, and particle diameter is smaller and nano-particle favorable dispersibility.
Embodiment nine
A kind of preparation method of polymer albumin nanospheres, comprises the following steps：
(1) the PSA aqueous solution that volume mass concentration is 200mg/mL (w/v) is taken, then using 10mol/L NaOH solution adjust the pH value of the albumin aqueous solution to 12；
(2) reaction solution is obtained to addition dithiothreitol (DTT) in the albumin aqueous solution that the pH value obtained by step (1) is 12, Then reaction 240 hours is shaken gently at 60 DEG C, in the reaction solution, the molal quantity of the dithiothreitol (DTT) is albumin 5000 times of molal quantity；
(3) step (2) reacted solution is carried out into ultrasound under conditions of 60 DEG C, ultrasonic power is 1KW, while in institute Stating is carried out injecting ethanol solution with the speed of 1000ml/s in ultrasonic solution, and dimethyl sulfoxide (DMSO) and volume matter are contained in ethanol solution Amount concentration is the chlorin e 6 of 0.1mg/mL, and the volume ratio of the etoh solvent in dimethyl sulfoxide (DMSO) and ethanol solution is 1:9, obtain To microemulsion solution, after the microemulsion solution reacts 5min under conditions of 60 DEG C, obtain outstanding containing polymer albumin nanospheres Turbid liquid；The volume that ethanol solution is added is 100 times of the step (2) reacted liquor capacity；
(4) suspension containing polymer albumin nanospheres obtained by step (3) is moved into bag filter, at room temperature, will be saturating Analysis bag is placed in dialysis 300 hours in the PBS that 5L pH are 12, and period every 12 hours change liquid 1 time, every time using 5L PH is 12 PBS, bag filter is placed in 5L distilled waters again then and is dialysed 12 hours, obtains polymer albumin nano Ball solution；
(5) pre-freeze is transferred to negative at the polymer albumin nanospheres solution obtained by step (4) being placed in into 0 DEG C after 1 hour Freezed 2 hours at 20 DEG C, then freeze-drying 72 hours in freeze drier, obtain polymer albumin nanospheres, described Target is contained in polymer albumin nanospheres and has delivered thing；It is chlorin e 6 that the target delivers thing；Polymer albumin Including the albumin molecule containing sulfydryl and/or disulfide bond, it is connected with each other by disulfide bond between albumin molecule, the white egg of polymer The particle diameter of white nanosphere is 300~400nm.
Embodiment ten
A kind of preparation method of polymer albumin nanospheres, comprises the following steps：
(1) the Recombinant Serum Albumin aqueous solution that volume mass concentration is 300mg/mL (w/v) is taken, is then used The NaOH solution of 0.1mol/L adjusts the pH value of the albumin aqueous solution to 9；
(2) reaction solution is obtained to addition beta -mercaptoethanol in the albumin aqueous solution that the pH value obtained by step (1) is 9, Then reaction 0.05 hour is shaken gently at 30 DEG C, in the reaction solution, the molal quantity of the beta -mercaptoethanol is white egg 100 times of white molal quantity；
(3) step (2) reacted solution is carried out into ultrasound under conditions of 30 DEG C, ultrasonic power is 50KW, while Ethanol solution is injected with the speed of 0.1ml/s in the solution for carrying out ultrasound, dimethyl sulfoxide (DMSO) and volume are contained in ethanol solution Mass concentration is the chlorin e 6 of 5mg/mL, and the volume ratio of the etoh solvent in dimethyl sulfoxide (DMSO) and ethanol solution is 1:9, obtain To microemulsion solution, after the microemulsion solution reacts 240min under conditions of 30 DEG C, the outstanding of polymer albumin nanospheres is obtained Turbid liquid；The volume that ethanol solution is added is 10 times of step (2) reacted liquor capacity；
(4) the polymer albumin nanospheres suspension obtained by step (3) is moved into bag filter, keeping temperature is 60 DEG C Under the conditions of, bag filter is placed in the PBS that 100mL pH are 7 and is dialysed 2 hours, it is 7 that 100mL pH are used every time PBS, period every 1 hour changes liquid 1 time, bag filter is placed in 100mL distilled waters again then and is dialysed 12 hours, obtains Polymer albumin nanospheres solution；
(5) pre-freeze is transferred to after 48 hours at the polymer albumin nanospheres solution obtained by step (4) being placed in into minus 4 DEG C Freezed 48 hours at minus 50 DEG C, then freeze-drying 96 hours in freeze drier, obtain polymer albumin nanospheres, institute State and contained in polymer albumin nanospheres target and deliver thing；It is chlorin e 6 that the target delivers thing；The white egg of polymer The white albumin molecule included containing sulfydryl and/or disulfide bond, is connected with each other between albumin molecule by disulfide bond, and polymer is white The particle diameter of protein nano ball is 300~500nm.
Embodiment 11
(1) take albumin mixed liquor, albumin mixed liquor contain the bovine serum albumin(BSA) that concentration is 100mg/mL (w/v), Concentration is the chlorin e 6 and dimethyl sulfoxide (DMSO) of 2mg/ml, the volume ratio of aqueous solvent in dimethyl sulfoxide (DMSO) and albumin mixed liquor It is 1:9, then using 0.1mol/L NaOH solution regulation mixed liquor pH value to 9；
(2) to adding glutathione to obtain reaction solution in step (1) albumin mixed liquor, then it is shaken gently at 30 DEG C anti- Answer 1 hour, in reaction solution, the molal quantity of glutathione is 1000 times of albumin molar number；
(3) step (2) reacted solution is carried out into ultrasound under conditions of 4 DEG C, ultrasonic power is 100KW, while will It is injected into absolute ethyl alcohol with the speed of 0.01mL/s in the solution for carrying out ultrasound, obtains microemulsion solution, the microemulsion solution is 4 After reacting 10min under conditions of DEG C, the suspension containing polymer albumin nanospheres is obtained；The volume that absolute ethyl alcohol is added is step Suddenly 50 times of (2) reacted liquor capacity；
(4) suspension containing polymer albumin nanospheres obtained by step (3) is moved into bag filter, keeping temperature is 0 Under conditions of DEG C, bag filter is placed in the PBS that 100mL pH are 7 and is dialysed 2 hours, period every 1 hour changes liquid 1 Secondary, it is 7 PBS that 100mL pH are used every time, bag filter is then placed in 100mL distilled waters into dialysis 12 again small When, obtain polymer albumin nanospheres solution；
(5) pre-freeze is transferred to after 2 hours at the polymer albumin nanospheres solution obtained by step (4) being placed in into minus 20 DEG C Freezed 24 hours at minus 80 DEG C, then freeze-drying 48 hours in freeze drier, obtain polymer albumin nanospheres, institute State and contained in polymer albumin nanospheres target and deliver thing；It is chlorin e 6 that the target delivers thing；The white egg of polymer The white albumin molecule included containing sulfydryl and/or disulfide bond, is connected with each other between albumin molecule by disulfide bond, and polymer is white The particle diameter of protein nano ball is 500~700nm.
Embodiment 12
(1) albumin mixed liquor is taken, concentration containing volume mass is the cow's serum of 100mg/mL (w/v) in albumin mixed liquor Albumin, concentration are the chlorin e 6 and dimethyl sulfoxide (DMSO) of 2mg/ml, aqueous solvent in dimethyl sulfoxide (DMSO) and albumin mixed liquor Volume ratio be 1:9, then using 0.1mol/L NaOH solution regulation mixed liquor pH value to 9；
(2) to adding glutathione to obtain reaction solution in step (1) gained albumin mixed liquor, then gently shaken at 30 DEG C Dynamic reaction 1 hour, in the reaction solution, the molal quantity of the glutathione is 100 times of albumin molar number；
(3) step (2) reacted solution is carried out into ultrasound under conditions of 0 DEG C, ultrasonic power is 10KW, while will enter It is injected into ethanol solution with the speed of 0.01mL/s in the solution of row ultrasound, the ethanol solution contains dimethyl sulfoxide (DMSO) and volume matter Amount concentration is the chlorin e 6 of 0.1mg/mL, and the volume ratio of the etoh solvent in dimethyl sulfoxide (DMSO) and ethanol solution is 1:9, obtain To microemulsion solution, after the microemulsion solution reacts 10min under conditions of 0 DEG C, obtain outstanding containing polymer albumin nanospheres Turbid liquid；The volume that ethanol solution is added is 20 times of step (2) reacted liquor capacity；
(4) suspension containing polymer albumin nanospheres obtained by step (3) is moved into bag filter, keeping temperature is 0 Under conditions of DEG C, bag filter is placed in the PBS that 100mL pH are 7 and is dialysed 2 hours, period every 1 hour changes liquid 1 Secondary, it is 7 PBS that 100mL pH are used every time, bag filter is then placed in 100mL distilled waters into dialysis 12 again small When, obtain polymer albumin nanospheres solution；
(5) pre-freeze is transferred to after 2 hours at the polymer albumin nanospheres solution obtained by step (4) being placed in into minus 20 DEG C Freezed 24 hours at minus 80 DEG C, then freeze-drying 48 hours in freeze drier, obtain polymer albumin nanospheres, institute State and contained in polymer albumin nanospheres target and deliver thing；It is chlorin e 6 that the target delivers thing；The white egg of polymer The white albumin molecule included containing sulfydryl and/or disulfide bond, is connected with each other, polymer albumin between albumin by disulfide bond The particle diameter of nanosphere is 700~1000nm.
Application Example
In order to verify that polymer albumin nanospheres provided in an embodiment of the present invention are preparing prevention, treatment or diagnosis cancer Medicine in application effect, Application Example have studied transmitting procedure of the polymer albumin nanospheres in Mice Body and right The specificity of breast tumor tissue identification and sensitivity, and it is white to set the polymer of different experimental group checking embodiments offers Protein nano ball suppresses the effect of tumour growth, as a result sees Fig. 8 and Fig. 9.
With MCF-7 tumor-bearing mices as model, the polymer albumin nanospheres water prepared by tail vein injection embodiment 8 Solution (concentration is 0.2mg/mL), then using using fluorescent vital imaging system (model MaestroTM2MaestroTM EX- RRO, company is U.S. CRi MaestroTM) and photoacoustic imaging platform (light source be tunable optical parametric oscillator, wave-length coverage It is 400nm-2500nm, model Vibrant355II HE, Opotek, Carlsbad, USA, light source pulse repetition rate 10Hz, Pulsewidth 5ns；Ultrasonic probe centre frequency 10MHz, model V315, Panametrics, Waltham, US) it is white to study polymer Transmitting procedure of the protein nano ball in Mice Body and the specificity to breast tumor tissue identification and sensitivity, Fig. 8 is real Polymer albumin nanospheres obtained in example 8 are applied for the fluorescence and photoacoustic imaging of tumor bearing nude mice tumor tissues (in dotted line frame) Figure；In Fig. 8, a and c figures do not inject the fluorescence imaging figure and photoacoustic imaging figure, b and d of polymer albumin nanospheres respectively Figure is respectively injection the fluorescence imaging figure and photoacoustic imaging figure of polymer albumin nanospheres, as can be seen from Figure 8, does not have Without fluorescence and photoacoustic signal, injection has polymer albumin nanospheres to tumor locus during injection polymer albumin nanospheres Tumor-bearing mice 24h after tumor locus fluorescence and photoacoustic signal be enhanced, illustrate polymer albumin nanospheres to tumour Tissue has very strong targeting.
Fig. 9 is the tumour growth that polymer albumin nanospheres obtained in embodiment 8 are used for tumor bearing nude mice light dynamic experiment Curve.Specific experiment scheme：By 30 nude mices (Balb/c) of 6~8 weeks, 6 one group is divided into 5 experimental groups and carries out entity tumor Modeling, including：1.PBS buffer solutions group (control group)；2. dissociating, (injection volume of Ce6 is 2mg/kg to Ce6, i.e., per 1kg mouse note Penetrate 2mg Ce6)+light group；3. (injection volume of Ce6 is the polymer albumin nanospheres containing Ce6 that prepared by embodiment 8 2mg/kg, i.e., polymer albumin nanospheres of the injection containing 2mg Ce6 per 1kg mouse)+not light group；4. prepared by embodiment 8 The albumin nanospheres of the polymer containing Ce6+light group (injection volume of Ce6 be 2mg/kg) 5. containing of preparing of embodiment 8 Polymer albumin nanospheres+the light group (injection volume of Ce6 is 1mg/kg) of Ce6.It is long to 100mm in tumor model3Afterwards, press Experimental program according to above-mentioned 5 experimental groups is tested, and is observed tumour growth situation and is drawn tumor growth curve.Using transmitting Wavelength is the near-infrared laser elective irradiation mouse tumor tissue of 660nm.
It can be seen in figure 9 that the volume growth rate of tumour is very fast in control group, and will be included obtained in embodiment 8 The polymer albumin nanospheres of Ce6 irradiate tumor tissues by tail vein injection to Mice Body and with near-infrared laser Gross tumor volume in experimental group increases to receive and necessarily suppresses, and as the concentration of Ce6 increases, tumor inhibitory effect is better.Phase For control group, the gross tumor volume of experimental group 4 reduces 70%.
To sum up, the present invention contains in hydrophilic polymer albumin hydrophobicity chlorin e 6, can improve two The water solubility of hydrogen porphines e6 (Ce6), while nanosphere of the present invention can be stable in the presence of in organism in blood circulation system, from And invalid release of the chlorin e 6 in blood circulation system transmitting procedure is avoided, polymer albumin nanospheres also have master Moving-target tropism, contributes to chlorin e 6 quickly and efficiently into cell to improve the curative effect of photodynamic therapy.Meanwhile, it is many Aggressiveness albumin nanospheres also have good near-infrared fluorescent and photoacoustic imaging ability, can be used in real time, non-invasively monitoring control Nano-particle conveying behavior in vivo, real-time assessment can be carried out after optical dynamic therapy by being imaged to curative effect before treating, and be implemented as As the photodynamic therapy of guiding.
Presently preferred embodiments of the present invention is the foregoing is only, is not intended to limit the invention, it is all in essence of the invention Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.

Claims (9)

1. a kind of preparation method of polymer albumin nanospheres, it is characterised in that comprise the following steps：
(1) prepare volume mass concentration and be the albumin aqueous solution containing sulfydryl and/or disulfide bond of 0.01~300mg/mL, and adjust The pH value for saving the albumin aqueous solution is 7~12；
Wherein, thing is delivered containing target in the albumin aqueous solution；
The target delivers thing includes cancer therapy drug；
The cancer therapy drug includes (2S- is trans) -18- carboxyls -20- (carboxymethyl) -13- ethyl -2,3- dihydros 3,7,12,17- Tetramethyl -8- vinyl -21H, 23H- porphines -2- propionic acid；
(2) to adding the reducing agent with sulfydryl must to react in the albumin aqueous solution that the pH value obtained by step (1) is 7~12 Liquid, is then shaken gently for reaction 0.05~12 hour at 0~60 DEG C, in the reaction solution, the reducing agent with sulfydryl Molal quantity be 10~5000 times of albumin molar number；
(3) step (2) reacted solution is carried out into ultrasound under conditions of 0~60 DEG C, the power bracket of the ultrasound for 1~ 100KW, while the speed addition with 50~1000mL/s in the solution for carrying out ultrasound under conditions of stirring is organic molten Agent obtains microemulsion solution, and after the microemulsion solution reacts 5~240min under conditions of 0~60 DEG C, then stratification removes Organic phase, obtains the suspension containing polymer albumin nanospheres, wherein, the volume that the organic solvent is added is the step (2) 1~100 times of reacted solution；
(4) in 0~60 DEG C and pH value it is 9~12 by the suspension containing polymer albumin nanospheres obtained by step (3) Under the conditions of dialysed, obtain polymer albumin nanospheres solution；
(5) the polymer albumin nanospheres solution obtained by step (4) is dried dewater treatment, obtains polymer albumin Nanosphere, the polymer albumin nanospheres include the albumin molecule containing sulfydryl and/or disulfide bond, the albumin molecule Between by disulfide bond be connected with each other,
Wherein, target has been contained in the polymer albumin nanospheres and has delivered thing；
The target delivers thing includes cancer therapy drug；
The cancer therapy drug includes (2S- is trans) -18- carboxyls -20- (carboxymethyl) -13- ethyl -2,3- dihydros 3,7,12,17- Tetramethyl -8- vinyl -21H, 23H- porphines -2- propionic acid；
The particle diameter of the polymer albumin nanospheres is 10~1000nm.
2. a kind of preparation method of polymer albumin nanospheres as claimed in claim 1, it is characterised in that the step (1) in, the albumin molecule containing sulfydryl and/or disulfide bond is human serum albumins, bovine serum albumin(BSA), the pure egg of pig blood In vain, at least one in Recombinant Serum Albumin and hemoglobin.
3. a kind of preparation method of polymer albumin nanospheres as claimed in claim 1, it is characterised in that the step (1) in, the quality that the target delivers thing is 0.0002~0.5 times of the albumin quality containing sulfydryl and/or disulfide bond.
4. a kind of preparation method of polymer albumin nanospheres as claimed in claim 1, it is characterised in that the step (2) in, the reducing agent with sulfydryl is glutathione, cysteine, mercaptoethanol or dithiothreitol (DTT).
5. a kind of preparation method of polymer albumin nanospheres as claimed in claim 1, it is characterised in that step (2) institute State reaction solution and reaction, the bar of step (2) reacted solution described in step (3) at 4~60 DEG C are shaken gently at 4~60 DEG C Ultrasound is carried out under part, the microemulsion solution reacts 5~240min under conditions of 4~60 DEG C, and step (4) is described white containing polymer The suspension of protein nano ball is dialysed at 4~60 DEG C.
6. a kind of preparation method of polymer albumin nanospheres as claimed in claim 1, it is characterised in that the step (3) in, the organic solvent is at least one in methyl alcohol, ethanol, propyl alcohol and the tert-butyl alcohol.
7. a kind of polymer albumin nanospheres, it is characterised in that be obtained according to preparation method as claimed in claim 1；Institute Stating polymer albumin nanospheres includes the albumin molecule containing sulfydryl and/or disulfide bond, passes through between the albumin molecule Disulfide bond is connected with each other,
Target is contained in the polymer albumin nanospheres and has delivered thing；
The target delivers thing includes cancer therapy drug；
The cancer therapy drug includes (2S- is trans) -18- carboxyls -20- (carboxymethyl) -13- ethyl -2,3- dihydros 3,7,12,17- Tetramethyl -8- vinyl -21H, 23H- porphines -2- propionic acid；
The particle diameter of the polymer albumin nanospheres is 10~1000nm.
8. a kind of polymer albumin nanospheres as claimed in claim 7, it is characterised in that described containing sulfydryl and/or two sulphur The albumin molecule of key is human serum albumins, bovine serum albumin(BSA), PSA, Recombinant Serum Albumin and blood red egg At least one in white.
9. polymer albumin nanospheres as claimed in claim 7 or 8 are in the medicine for preparing prevention, treatment or diagnosis cancer Application.
CN201410337774.3A 2013-09-27 2014-07-16 A kind of polymer albumin nanospheres comprising chlorin e 6 and its preparation method and application Active CN104162171B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201410337774.3A CN104162171B (en) 2013-09-27 2014-07-16 A kind of polymer albumin nanospheres comprising chlorin e 6 and its preparation method and application

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201310449770.XA CN103495179A (en) 2013-09-27 2013-09-27 Polymer albumin nanosphere as well as preparation method and applications of nanosphere
CN201310449770X 2013-09-27
CN201310449770.X 2013-09-27
CN201410337774.3A CN104162171B (en) 2013-09-27 2014-07-16 A kind of polymer albumin nanospheres comprising chlorin e 6 and its preparation method and application

Publications (2)

Publication Number Publication Date
CN104162171A CN104162171A (en) 2014-11-26
CN104162171B true CN104162171B (en) 2017-06-23

Family

ID=49860493

Family Applications (6)

Application Number Title Priority Date Filing Date
CN201310449770.XA Pending CN103495179A (en) 2013-09-27 2013-09-27 Polymer albumin nanosphere as well as preparation method and applications of nanosphere
CN201410337774.3A Active CN104162171B (en) 2013-09-27 2014-07-16 A kind of polymer albumin nanospheres comprising chlorin e 6 and its preparation method and application
CN201410337773.9A Active CN104162164B (en) 2013-09-27 2014-07-16 A kind of polymer albumin nanospheres comprising indocyanine-green and its preparation method and application
CN201410338871.4A Pending CN104162165A (en) 2013-09-27 2014-07-16 Lymphatic tracer-containing polymer albumin nanosphere and preparation method and application thereof
CN201410337805.5A Active CN104189916B (en) 2013-09-27 2014-07-16 Multimer alhumin nanospheres, and preparation method and application thereof
CN201410338882.2A Active CN104162172B (en) 2013-09-27 2014-07-16 A kind of polymer albumin nanospheres comprising taxol and its preparation method and application

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN201310449770.XA Pending CN103495179A (en) 2013-09-27 2013-09-27 Polymer albumin nanosphere as well as preparation method and applications of nanosphere

Family Applications After (4)

Application Number Title Priority Date Filing Date
CN201410337773.9A Active CN104162164B (en) 2013-09-27 2014-07-16 A kind of polymer albumin nanospheres comprising indocyanine-green and its preparation method and application
CN201410338871.4A Pending CN104162165A (en) 2013-09-27 2014-07-16 Lymphatic tracer-containing polymer albumin nanosphere and preparation method and application thereof
CN201410337805.5A Active CN104189916B (en) 2013-09-27 2014-07-16 Multimer alhumin nanospheres, and preparation method and application thereof
CN201410338882.2A Active CN104162172B (en) 2013-09-27 2014-07-16 A kind of polymer albumin nanospheres comprising taxol and its preparation method and application

Country Status (1)

Country Link
CN (6) CN103495179A (en)

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104043135B (en) * 2014-06-13 2017-05-24 苏州大学 Albumin indocyanine green paclitaxel compound as well as preparation method and application thereof
CN105288622B (en) * 2015-11-03 2018-06-19 浙江大学 The preparation method of chemotherapeutics and the cell membrane vesicles of optical dynamic therapy medicine is loaded simultaneously
CN106310290B (en) * 2016-10-27 2019-08-23 深圳先进技术研究院 A kind of tumor-targeting temperature-sensitive prodrug and the preparation method and application thereof
CN108295046A (en) * 2016-12-30 2018-07-20 中国科学院深圳先进技术研究院 The preparation method and albumin nanoparticle obtained of a kind of albumin nanoparticle and application
CN108283487A (en) * 2017-01-10 2018-07-17 衍全生物科技（太仓）有限公司 Cancer photo-thermal ultrasonography device and technology
CN107028898B (en) * 2017-06-14 2021-05-04 中国人民解放军军事医学科学院毒物药物研究所 Irinotecan medicine freeze-dried preparation, and preparation method and application thereof
CN108379228B (en) * 2018-02-28 2021-02-23 湖南大学 Albumin nano-particles wrapping pharmacological active substances and preparation method and application thereof
CN109157662B (en) * 2018-06-06 2021-07-20 北京大学 Human serum albumin-adriamycin cross-linked substance nano-particles and application thereof
CN110201191B (en) * 2019-07-10 2022-03-25 莎穆(上海)生物科技有限公司 Compound of functional protein and cyanine dye molecule and preparation method and application thereof
CN110403916B (en) * 2019-07-31 2021-08-10 深圳大学 Nano therapeutic agent and preparation method and application thereof
CN111632144B (en) * 2020-07-06 2022-07-29 聊城大学 High-drug-loading-rate photo-chemotherapy bifunctional nanoparticles and preparation method thereof
CN111840549B (en) * 2020-07-22 2022-11-04 苏州大学 Platinum drug/photosensitizer-loaded protein nanoparticles and preparation method and application thereof
WO2022016555A1 (en) * 2020-07-24 2022-01-27 苏州大学 Platinum-based drug-/photosensitizer-loaded protein nanoparticle, and preparation method therefor and application thereof
CN112546220A (en) * 2020-12-09 2021-03-26 广东医科大学 Preparation method and application of oxygen-carrying hybrid protein-loaded metal complex nano system
CN112546221B (en) * 2020-12-11 2023-12-19 深圳先进技术研究院 Tumor diagnosis and treatment medicine and preparation method and application thereof
CN114948784A (en) * 2022-04-20 2022-08-30 重庆医科大学 Bionic nano-particles and preparation method and application thereof
CN115887677B (en) * 2022-11-03 2024-04-12 嘉兴学院 Preparation method and application of protein bionic capsules

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102048695A (en) * 2009-08-11 2011-05-11 南京大学 Preparation method of protein nanoparticle for in vivo delivery of pharmacologically active agent
EP2605019A2 (en) * 2010-08-13 2013-06-19 National Cancer Center Sentinel lymph node marker capable of multi-mode imaging
CN103169968A (en) * 2013-03-12 2013-06-26 中国科学院理化技术研究所 Hydrophobic dihydroporphin photosensitizer nanometer pharmaceutic preparation based on albumin as well as preparation method and application of nanometer pharmaceutic preparation
CN103212083A (en) * 2012-12-19 2013-07-24 清华大学 Method for preparing stable albumin nano-particles

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101385857A (en) * 2008-10-21 2009-03-18 中国药科大学 Novel nano preparation with stable protein and preparation method and use thereof
KR101043407B1 (en) * 2009-02-19 2011-06-22 한국과학기술연구원 A tumor targeting protein conjugate and a method for preparing the same

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102048695A (en) * 2009-08-11 2011-05-11 南京大学 Preparation method of protein nanoparticle for in vivo delivery of pharmacologically active agent
EP2605019A2 (en) * 2010-08-13 2013-06-19 National Cancer Center Sentinel lymph node marker capable of multi-mode imaging
CN103212083A (en) * 2012-12-19 2013-07-24 清华大学 Method for preparing stable albumin nano-particles
CN103169968A (en) * 2013-03-12 2013-06-26 中国科学院理化技术研究所 Hydrophobic dihydroporphin photosensitizer nanometer pharmaceutic preparation based on albumin as well as preparation method and application of nanometer pharmaceutic preparation

Also Published As

Publication number Publication date
CN104189916A (en) 2014-12-10
CN104162165A (en) 2014-11-26
CN104162171A (en) 2014-11-26
CN104162172A (en) 2014-11-26
CN104162164B (en) 2017-06-23
CN104162164A (en) 2014-11-26
CN104189916B (en) 2017-05-10
CN104162172B (en) 2017-06-23
CN103495179A (en) 2014-01-08

Similar Documents

Publication Publication Date Title
CN104162171B (en) A kind of polymer albumin nanospheres comprising chlorin e 6 and its preparation method and application
US20240042063A1 (en) LINKED AND OTHER pH-TRIGGERED COMPOUNDS
Liu et al. Combined photothermal and photodynamic therapy delivered by PEGylated MoS 2 nanosheets
Gao et al. Near-infrared light-triggered switchable nanoparticles for targeted chemo/photothermal cancer therapy
Wang et al. Integration of cascade delivery and tumor hypoxia modulating capacities in core-releasable satellite nanovehicles to enhance tumor chemotherapy
Wang et al. Central metal-derived co-assembly of biomimetic GdTPP/ZnTPP porphyrin nanocomposites for enhanced dual-modal imaging-guided photodynamic therapy
WO2015188570A1 (en) Albumin indocyanine green and paclitaxel compound and preparation method and use thereof
CN103623430B (en) Preparation method and application of targeted co-supported drug delivery system nano-particles based on polylactic-co-glycolic acid
CA2516116A1 (en) Nanoparticle based stabilization of ir fluorescent dyes
Chen et al. Glucosamine derivative modified nanostructured lipid carriers for targeted tumor delivery
ChingáGoh et al. Polymeric nanorods with aggregation-induced emission characteristics for enhanced cancer targeting and imaging
Du et al. Intracellular tracking of drug release from pH-sensitive polymeric nanoparticles via FRET for synergistic chemo-photodynamic therapy
CN105968372B (en) A kind of autofluorescence nanogel and the preparation method and application thereof
Yang et al. NIR-activated self-sensitized polymeric micelles for enhanced cancer chemo-photothermal therapy
Liu et al. NIRF/PA/CT multi-modality imaging guided combined photothermal and photodynamic therapy based on tumor microenvironment-responsive nanocomposites
Han et al. Acidity-triggered tumor retention/internalization of chimeric peptide for enhanced photodynamic therapy and real-time monitoring of therapeutic effects
CN106581693A (en) Application of ursolic acid conjugate having anticancer activity and serving as drug carrier or molecular probe carrier
Yang et al. Self-assembled multifunctional polymeric micelles for tumor-specific bioimaging and synergistic chemo-phototherapy of cancer
Lee et al. Hybrid nanoparticles based on chlorin e6-conjugated hyaluronic acid/poly (l-histidine) copolymer for theranostic application to tumors
Stromberg et al. Formulation of stabilizer-free, nontoxic PLGA and elastin-PLGA nanoparticle delivery systems
CN103965420B (en) A kind of based on disulfide bond reduction response type degradable medicaments carrier and preparation and application
CN106166141A (en) A kind of Multifunctional composite nanometer medicine for tumor imaging and treatment and preparation method thereof
Yang et al. A cyanine-based polymeric nanoplatform with microenvironment-driven cascaded responsiveness for imaging-guided chemo-photothermal combination anticancer therapy
Zeng et al. Highly efficient Chemo/Photothermal therapy alleviating tumor hypoxia against cancer and attenuate liver metastasis in vivo
Zhu et al. Self-Assembly of Precisely Fluorinated Albumin for Dual Imaging-Guided Synergistic Chemo–Photothermal–Photodynamic Cancer Therapy

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant