CN107669658A - A kind of load enzyme nanometer microvesicle of folacin receptor targeting and preparation method thereof - Google Patents
A kind of load enzyme nanometer microvesicle of folacin receptor targeting and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of load enzyme nanometer microvesicle of folacin receptor targeting, the load enzyme nanometer microvesicle is made up of kernel templates and shell, and kernel templates are the compound of CdSe/ZnS quantum dot coelenterons luciferase 8；Shell is folic acid cross-linked polyethylene glycol polylactic-co-glycolic acid polymer.The invention also discloses the preparation method of the load enzyme nanometer microvesicle.The load enzyme nanometer microvesicle of the present invention has Targeting delivery, delays to degrade, kept for the advantages of activity in vivo of coelenteron luciferase 8.
Description
Technical field
The present invention relates to pharmaceutical activity protective agent field and target medicine carrier synthesis field, and in particular to a kind of folic acid by
Load enzyme nanometer microvesicle of body targeting and preparation method thereof.
Background technology
Optical dynamic therapy (Photodynamic Therapy, PDT) is that the one of tumour is treated after operation, chemotherapy, radiotherapy
Kind new method.Good therapeutic effect is obtained in the superficial tissues such as cutaneum carcinoma, breast cancer tumour.At present, optical dynamic therapy is main
Using generating laser as external light source, the sensitising agent by being injected intravenously and being distributed in tumor tissues is excited
(Photosensitizer, PS) so as to trigger photodynamic reaction (Photodaynamic Reaction, PDR) produce cell toxicant
Agent-active oxygen (Reactive Oxygen Species, ROS), causes tumor cell necrosis and/or apoptosis, and destroys tumour week
New vessels is enclosed, and then kills tumor tissues
Because the exogenous laser penetration power used in optical dynamic therapy is weaker, deep tissue can not be acted on and trigger light
Dynamic response, therefore, optical dynamic therapy are restricted in the treatment of the deep tissues such as liver cancer.
Enzymatic in biosystem is luminous to be referred to as bioluminescence.This is one general in bacterium, algae, insect etc.
All over existing phenomenon.Bioluminescence is typically derived from enzyme (such as luciferase) catalysis substrate (such as fluorescein) and oxidation reaction occurs,
Discharge light quantum.Based on this principle, in recent years, it is deep in optical dynamic therapy liver cancer etc. that researchers have started bio-luminescence system
Research in portion's tissue, that is, by the way that enzyme-to-substrate is delivered at tumour, lighted, there is provided light source.However, enzyme enters
Easily it is degraded quickly after in vivo, activity can not be ensured.
In addition, optical dynamic therapy is also a problem urgently to be resolved hurrily in the targeting of tumour, because existing smooth power is controlled
The research for rarely having and being related to the deep tissues such as liver is treated, optical dynamic therapy is referred from and applies to other superficial tissues, its targeting master
It is fixed against targeting irradiation of the tumor tissues to this passive target effect of the retention effect of sensitising agent, and manipulation light source.This
The relatively low passive target of species specificity, easily causes the damage of normal structure, hinders the further utilization of optical dynamic therapy, because
How this, ensure the targeting of enzyme, and a problem.
The content of the invention
For defect present in prior art, it is an object of the invention to provide a kind of load enzyme of folacin receptor targeting to receive
Rice microvesicle, the load enzyme nanometer microvesicle have the advantages of Targeting delivery is with degraded is delayed, and keep coelenteron 8 work in vivo of luciferase
Property.
To achieve the above objectives, the present invention adopts the technical scheme that：A kind of load enzyme nanometer microvesicle of folacin receptor targeting,
The load enzyme nanometer microvesicle is made up of kernel templates and shell, and the kernel templates are CdSe/ZnS quantum dots-coelenteron fluorescein
The compound of enzyme 8；The shell is folic acid cross-linked polyethylene glycol polylactic-co-glycolic acid polymer.
Further, the CdSe/ZnS quantum dots-compound of coelenteron luciferase 8 is by CdSe/ZnS quantum dots and enteric cavity
The covalent coupling of luciferase 8 forms.
Present invention also offers a kind of preparation method of the load enzyme nanometer microvesicle of folacin receptor targeting as described above, it is wrapped
Include following steps：
S1：The folic acid cross-linked polyethylene glycol polylactic-co-glycolic acid polymer is prepared, and forms it into the shell；
S2：The CdSe/ZnS quantum dots-compound of coelenteron luciferase 8 is prepared, and forms it into the kernel templates；
S3：The kernel templates are loaded into the shell, complete the preparation for carrying enzyme nanometer microvesicle.
Further, kernel templates preparation method described in S2 are as follows：
1~3ml cushioning liquid is added in glutaraldehyde, and adds 8~12 μ l CdSe/ZnS quantum dots, in argon gas ring
After stirring 1.5~2.5h in border, separating-purifying simultaneously removes glutaraldehyde；1~3mg coelenterons luciferase 8 is added, is continued in argon gas ring
In border after 9~11h of stirring reaction, separating-purifying removes coelenteron luciferase 8, and it is glimmering to obtain the CdSe/ZnS quantum dots-coelenteron
The compound of light element enzyme 8.
Further, the cushioning liquid is borate buffer solution or citrate buffer solution.
Further, the pH value of the cushioning liquid is 7.2~7.6.
Further, the preparation method that the kernel templates are loaded into the shell in S3 is as follows：
S31：The kernel templates and water are mixed to form interior aqueous phase, by interior aqueous phase be added to the shell concentration for 0~
In 0.050g/ml dichloromethane, colostric fluid is obtained；
S32：The shell and water are mixed to form outer aqueous phase, colostric fluid is distributed to outer aqueous phase using SPG membrane emulsifiers
In, obtain double emulsion；
S33：Double emulsion is transferred in magnetic stirring apparatus, stirring dichloromethane is volatilized, stir speed (S.S.) be 80~
120rpm/min, whipping temp are 30~50 DEG C, and mixing time is 8~10h；
S34：Double emulsion is transferred in centrifuge, precipitation is collected by centrifugation under the conditions of 3~5 DEG C, 2500~3500g, is done
It is dry.
Further, interior aqueous phase is at the uniform velocity added in dichloromethane using syringe.
Further, pressure is 45~55kPa in SPG membrane emulsifiers pressure chamber, and colostric fluid is at the uniform velocity distributed to by SPG films
In outer aqueous phase.
Compared with prior art, the advantage of the invention is that：
(1) present invention has wrapped up crust for coelenteron luciferase 8, can improve to the activity of coelenteron luciferase 8
Protection, prevents it to be degraded before the deep tissues such as liver are reached, ensure that bioactivity and stability.
(2) case surface of the present invention is modified with folic acid, can realize active targeting with the folacin receptor of the high expression of liver cancer cells,
Accurate positioning；Meanwhile the shell can be played after the enrichment of coelenteron luciferase 8, carry out concentrating the effect for exciting and discharging, should
Shell is as one layer of lipid film, it is easier to penetrates tissue and cell membrane.
(3) load enzyme nanometer microvesicle prepared by the present invention has envelop rate stable, and uniform particle diameter, yield is high, synthesizes processing side
Method is simple, no biotoxicity.
Brief description of the drawings
Fig. 1 is load enzyme nanometer microbubble structure schematic diagram provided in an embodiment of the present invention.
Embodiment
The present invention is described in further detail below in conjunction with drawings and Examples.
Carry enzyme nanometer microvesicle embodiment 1：
Shown in Figure 1, the present invention provides a kind of load enzyme nanometer microvesicle of folacin receptor targeting, the load enzyme nanometer microvesicle by
Kernel templates and shell are formed, and the kernel templates are CdSe/ZnS quantum dots-compound of coelenteron luciferase 8, the CdSe/
ZnS quantum dot-compound of coelenteron luciferase 8 is formed by CdSe/ZnS quantum dots and the covalent coupling of enteric cavity luciferase 8；It is described
Shell is folic acid cross-linked polyethylene glycol polylactic-co-glycolic acid polymer.
Because in the presence of without CdSe/ZnS quantum dots, coelenteron luciferase 8 is catalyzed coelenteron fluorescein and oxidation reaction occurs
What is sent is 450~480nm of wavelength or so blue wave band, and the absorbing wavelength corresponding to sensitising agent is mostly 650nm's or so
Red spectral band, therefore, it is coupled using CdSe/ZnS quantum dots with coelenteron luciferase 8 compound, CdSe/ZnS quantum dots can rise
To the effect of Wavelength-converting, 450~480nm of absorbing wavelength or so blue light and the feux rouges for being immediately converts into 650nm or so, namely
Bioluminescence resonance energy transfer effect (Bioluminescence Resonance Energy Transfer).
The present invention has wrapped up crust for coelenteron luciferase 8, can improve the guarantor to the activity of coelenteron luciferase 8
Shield, prevents it to be degraded before the deep tissues such as liver are reached, ensure that bioactivity and stability；Meanwhile the shell can be with
Play after the enrichment of coelenteron luciferase 8, carry out concentrating the effect for exciting release；Case surface is modified with folic acid, can be with liver cancer
The folacin receptor of the high expression of cell realizes active targeting, accurate positioning, then concentrates release by ultrasound targeting blasting technique, can
The bio-light for producing sufficient intensity is used to excite sensitising agent, realizes that drug targeting delivers；The shell is as one layer of lipid film, no life
Thing toxicity, it is easier to penetrate tissue and cell membrane.
Preparation method embodiment 1：
The invention provides the preparation method of the load enzyme nanometer microvesicle of folacin receptor targeting, this method is preparing load enzyme nanometer
In microvesicle, there is the advantages of envelop rate stabilization, uniform particle diameter, yield are high, synthesis processing method is simple, it comprises the following steps：
S1：The folic acid cross-linked polyethylene glycol polylactic-co-glycolic acid polymer is prepared, and forms it into the shell；
S2：The CdSe/ZnS quantum dots-compound of coelenteron luciferase 8 is prepared, and forms it into the kernel templates；
S3：The kernel templates are loaded into the shell, complete the preparation for carrying enzyme nanometer microvesicle.
Preparation method embodiment 2：
The present embodiment and the difference of preparation method embodiment 1 be, in step sl, shell use molecular weight for 25KD,
Polylactic acid/hydroxy acetic acid (50 of the surface with carboxyl:50, it is designated as PLGA-COOH) it is carrier, the polyethylene glycol (molecule of double amino
Measure as 3.5KD, be designated as NH2-PEG-NH2) it is attachment, folic acid (being designated as FA) is specific targeting ligand.Using five-step approach system
PEG-PLA/glycolic acid polymer (being designated as PLGA-PEG-FA) of standby folate-targeted：
The first step：Synthesize PLGA-NHS：PLGA-COOH and n-hydroxysuccinimide (being designated as NHS) couple activation carboxyl.
Take 0.04MM PLGA-COOH to be dissolved in 4ml dichloromethane, take 0.4MM NHS and 0.3MM dicyclohexyl
Carbodiimide is dissolved in 2ml dichloromethane, the two mixing, is placed in shaking table and is shaken up at a slow speed overnight, add 80ml ice methanol
(methanol and ether volume ratio are 1 with ether mixed solution:1) shake up, 4 DEG C stand overnight, and see to have largely being settled out current, centrifugation
Collect precipitation, vacuum drying, you can obtain PLGA-NHS.
Second step：Synthesize the NH of mono amino protection2-PEG-NH-BOC：NH2-PEG-NH2(it is designated as with di-tert-butyl dicarbonate
BOC) react.
Take NH2-PEG-NH2It is dissolved in the sodium bicarbonate solution that pH is 7.40~7.50, final concentration of 1mg/ml；Take two
Dimethyl dicarbonate butyl ester is dissolved in 10ml dimethyl sulfoxide (DMSO)s, final concentration of 0.1mg/ml.By NH2-PEG-NH2With two dimethyl dicarbonate fourths
Ester mass ratio is 5:1 mixing, reaction is stirred at room temperature overnight, upper chromatographic column 15Q is isolated and purified after di-tert-butyl dicarbonate, is obtained
NH2-PEG-NH-BOC。
3rd step：Synthesize PLGA-PEG-NH-BOC：NH2- PEG-NH-BOC and PLGA-NHS coupling reactions.
Take 0.02MM PLGA-NHS to be dissolved in 20ml dichloromethane, 0.05MM NH is added with this2-PEG-NH-
BOC and 0.4MM DIPEA, it is placed in shaking table and shakes up at a slow speed overnight, adds 80ml ice methanol and ether mixes
Closing solution, (methanol and ether volume ratio are 1:1), 4 DEG C stand overnight, and see to have largely being settled out now, precipitation are collected by centrifugation, very
Sky is dried, you can obtains PLGA-PEG-NH-BOC.
4th step：Synthesize PLGA-PEG-NH2：BOC in trifluoroacetic acid (being designated as TFA) displacement PLGA-PEG-NH-BOC.
Take 250mg PLGA-PEG-NH-BOC to be dissolved in 25ml100% trifluoroacetic acids, 50 times are added after being deprotected 30min
Distilled water terminating reaction, anti-phase purifying on the deprotection solution after terminating reaction, obtains PLGA-PEG-NH2Monomer；Add 5
(methanol and ether volume ratio are 1 to the ice methanol and ether mixed solution of times volume:1), 4 DEG C stand overnight, and seeing has a large amount of precipitations
During appearance, precipitation is collected by centrifugation, is dried in vacuo, you can obtain PLGA-PEG-NH2Dry powder.
5th step：PLGA-PEG-FA：PLGA-PEG-NH2It is coupled with folic acid (being designated as FA)
100ml PLGA-PEG-NH is taken respectively250ml 100% dimethyl sulfoxide, final concentration 2mg/ are dissolved in folic acid
ml.The two adds 6MM dicyclohexylcarbodiimide after mixing, be placed in shaking table and shake up at a slow speed overnight, 5 times of volumes of addition
(methanol and ether volume ratio are 1 for ice methanol and ether mixed solution:1), 4 DEG C stand overnight, see have it is a large amount of be settled out it is current, from
The heart collects precipitation, vacuum drying, you can obtain PLGA-PEG-FA.
Preparation method embodiment 3：
The present embodiment and the difference of preparation method embodiment 1 are that in step s 2, kernel templates use CdSe/ZnS amounts
Son point and the covalent coupling of coelenteron luciferase 8, are prepared using Euplotes woodruffi：
1ml cushioning liquid is added in glutaraldehyde, and adds 8 μ l CdSe/ZnS quantum dots, is stirred in ar gas environment
After 2.5h, separating-purifying simultaneously removes glutaraldehyde；2mg coelenterons luciferase 8 is added, continues the stirring reaction 11h in ar gas environment
Afterwards, separating-purifying removes coelenteron luciferase 8, obtains CdSe/ZnS quantum dots-compound of coelenteron luciferase 8, wherein, it is described
Cushioning liquid is the borate buffer solution or citrate buffer solution that pH value is 7.4.
Preparation method embodiment 4：
The present embodiment and the difference of preparation method embodiment 1 are that in step s 2, kernel templates use CdSe/ZnS amounts
Son point and the covalent coupling of coelenteron luciferase 8, are prepared using Euplotes woodruffi：
2ml cushioning liquid is added in glutaraldehyde, and adds 10 μ l CdSe/ZnS quantum dots, is stirred in ar gas environment
After mixing 1.5h, separating-purifying simultaneously removes glutaraldehyde；1mg coelenterons luciferase 8 is added, continues the stirring reaction in ar gas environment
After 10h, separating-purifying removes coelenteron luciferase 8, obtains CdSe/ZnS quantum dots-compound of coelenteron luciferase 8, wherein,
The cushioning liquid is the borate buffer solution or citrate buffer solution that pH value is 7.2.
Preparation method embodiment 5：
The present embodiment and the difference of preparation method embodiment 1 are that in step s 2, kernel templates use CdSe/ZnS amounts
Son point and the covalent coupling of coelenteron luciferase 8, are prepared using Euplotes woodruffi：
3ml cushioning liquid is added in glutaraldehyde, and adds 12 μ l CdSe/ZnS quantum dots, is stirred in ar gas environment
After mixing 2h, separating-purifying simultaneously removes glutaraldehyde；4mg coelenterons luciferase 8 is added, continues the stirring reaction 9h in ar gas environment
Afterwards, separating-purifying removes coelenteron luciferase 8, obtains CdSe/ZnS quantum dots-compound of coelenteron luciferase 8, wherein, it is described
Cushioning liquid is the borate buffer solution or citrate buffer solution that pH value is 7.6.
Preparation method embodiment 6：
The present embodiment and the difference of preparation method embodiment 1 are, in step s3, using supersound method combination mocromembrane
Emulsifier, the kernel templates are loaded into the shell to prepare the load enzyme nanometer microvesicle.
S31：The kernel templates of above-mentioned preparation and water are mixed to form interior aqueous phase, by interior aqueous phase by syringe at the uniform velocity
It is added in dichloromethane, that is, now the shell concentration is 0g/ml dichloromethane, the colostric fluid stablized；
S32：The shell of above-mentioned preparation and water are mixed to form outer aqueous phase, divided colostric fluid using SPG membrane emulsifiers
It is scattered in outer aqueous phase, obtains double emulsion；In this course, ensure that pressure is 45kPa in SPG membrane emulsifier pressure chambers, colostrum
Liquid is at the uniform velocity distributed in outer aqueous phase by SPG films；
S33：When outer aqueous phase engenders milky turbidity emulsion after no longer deepening, double emulsion is transferred into magnetic force stirs
Mix in device, stirring makes dichloromethane volatilize, stir speed (S.S.) 80rpm/min, and whipping temp is 30 DEG C, mixing time 8h；
S34：Double emulsion is transferred in centrifuge, precipitation is collected by centrifugation under the conditions of 3 DEG C, 2500g, distillation water washing is more
After secondary, vacuum freeze drying, water and dichloromethane is distilled completely, it is micro- to obtain load enzyme nanometer described in dry white powder
Bubble.
Preparation method embodiment 7：
The present embodiment and the difference of preparation method embodiment 1 are, in step s3, using supersound method combination mocromembrane
Emulsifier, the kernel templates are loaded into the shell to prepare the load enzyme nanometer microvesicle.
S31：The kernel templates of above-mentioned preparation and water are mixed to form interior aqueous phase, by interior aqueous phase by syringe at the uniform velocity
It is added in the dichloromethane that the shell concentration is 0.050g/ml, the colostric fluid stablized；
S32：The shell of above-mentioned preparation and water are mixed to form outer aqueous phase, divided colostric fluid using SPG membrane emulsifiers
It is scattered in outer aqueous phase, obtains double emulsion；In this course, ensure that pressure is 50kPa in SPG membrane emulsifier pressure chambers, colostrum
Liquid is at the uniform velocity distributed in outer aqueous phase by SPG films；
S33：When outer aqueous phase engenders milky turbidity emulsion after no longer deepening, double emulsion is transferred into magnetic force stirs
Mix in device, stirring makes dichloromethane volatilize, stir speed (S.S.) 110rpm/min, and whipping temp is 40 DEG C, mixing time 10h；
S34：Double emulsion is transferred in centrifuge, precipitation is collected by centrifugation under the conditions of 4 DEG C, 3000g, distillation water washing is more
After secondary, vacuum freeze drying, water and dichloromethane is distilled completely, it is micro- to obtain load enzyme nanometer described in dry white powder
Bubble.
Preparation method embodiment 8：
The present embodiment and the difference of preparation method embodiment 1 are, in step s3, using supersound method combination mocromembrane
Emulsifier, the kernel templates are loaded into the shell to prepare the load enzyme nanometer microvesicle.
S31：The kernel templates of above-mentioned preparation and water are mixed to form interior aqueous phase, by interior aqueous phase by syringe at the uniform velocity
It is added in the dichloromethane that the shell concentration is 0.025g/ml, the colostric fluid stablized；
S32：The shell of above-mentioned preparation and water are mixed to form outer aqueous phase, divided colostric fluid using SPG membrane emulsifiers
It is scattered in outer aqueous phase, obtains double emulsion；In this course, ensure that pressure is 55kPa in SPG membrane emulsifier pressure chambers, colostrum
Liquid is at the uniform velocity distributed in outer aqueous phase by SPG films；
S33：When outer aqueous phase engenders milky turbidity emulsion after no longer deepening, double emulsion is transferred into magnetic force stirs
Mix in device, stirring makes dichloromethane volatilize, stir speed (S.S.) 100rpm/min, and whipping temp is 40 DEG C, mixing time 9h；
S34：Double emulsion is transferred in centrifuge, precipitation is collected by centrifugation under the conditions of 4 DEG C, 3000g, distillation water washing is more
After secondary, vacuum freeze drying, water and dichloromethane is distilled completely, it is micro- to obtain load enzyme nanometer described in dry white powder
Bubble.
Preparation method embodiment 9：
The present embodiment and the difference of preparation method embodiment 1 are, in step s3, using supersound method combination mocromembrane
Emulsifier, the kernel templates are loaded into the shell to prepare the load enzyme nanometer microvesicle.
S31：The kernel templates of above-mentioned preparation and water are mixed to form interior aqueous phase, by interior aqueous phase by syringe at the uniform velocity
It is added in the dichloromethane that the shell concentration is 0.030g/ml, the colostric fluid stablized；
S32：The shell of above-mentioned preparation and water are mixed to form outer aqueous phase, divided colostric fluid using SPG membrane emulsifiers
It is scattered in outer aqueous phase, obtains double emulsion；In this course, ensure that pressure is 52kPa in SPG membrane emulsifier pressure chambers, colostrum
Liquid is at the uniform velocity distributed in outer aqueous phase by SPG films；
S33：When outer aqueous phase engenders milky turbidity emulsion after no longer deepening, double emulsion is transferred into magnetic force stirs
Mix in device, stirring makes dichloromethane volatilize, stir speed (S.S.) 120rpm/min, and whipping temp is 50 DEG C, mixing time 9h；
S34：Double emulsion is transferred in centrifuge, precipitation is collected by centrifugation under the conditions of 5 DEG C, 3000g, distillation water washing is more
After secondary, vacuum freeze drying, water and dichloromethane is distilled completely, it is micro- to obtain load enzyme nanometer described in dry white powder
Bubble.
Preparation method embodiment 10：
The present embodiment and the difference of preparation method embodiment 1 are, in step s3, using supersound method combination mocromembrane
Emulsifier, the kernel templates are loaded into the shell to prepare the load enzyme nanometer microvesicle.
S31：The kernel templates of above-mentioned preparation and water are mixed to form interior aqueous phase, by interior aqueous phase by syringe at the uniform velocity
It is added in the dichloromethane that the shell concentration is 0.025g/ml, the colostric fluid stablized；
S32：The shell of above-mentioned preparation and water are mixed to form outer aqueous phase, divided colostric fluid using SPG membrane emulsifiers
It is scattered in outer aqueous phase, obtains double emulsion；In this course, ensure that pressure is 48kPa in SPG membrane emulsifier pressure chambers, colostrum
Liquid is at the uniform velocity distributed in outer aqueous phase by SPG films；
S33：When outer aqueous phase engenders milky turbidity emulsion after no longer deepening, double emulsion is transferred into magnetic force stirs
Mix in device, stirring makes dichloromethane volatilize, stir speed (S.S.) 100rpm/min, and whipping temp is 47 DEG C, mixing time 8h；
S34：Double emulsion is transferred in centrifuge, precipitation is collected by centrifugation under the conditions of 4 DEG C, 3200g, distillation water washing is more
After secondary, vacuum freeze drying, water and dichloromethane is distilled completely, it is micro- to obtain load enzyme nanometer described in dry white powder
Bubble.
Preparation method embodiment 11：
The present embodiment and the difference of preparation method embodiment 1 are, in step s3, using supersound method combination mocromembrane
Emulsifier, the kernel templates are loaded into the shell to prepare the load enzyme nanometer microvesicle.
S31：The kernel templates of above-mentioned preparation and water are mixed to form interior aqueous phase, by interior aqueous phase by syringe at the uniform velocity
It is added in the dichloromethane that the shell concentration is 0.015g/ml, the colostric fluid stablized；
S32：The shell of above-mentioned preparation and water are mixed to form outer aqueous phase, divided colostric fluid using SPG membrane emulsifiers
It is scattered in outer aqueous phase, obtains double emulsion；In this course, ensure that pressure is 48kPa in SPG membrane emulsifier pressure chambers, colostrum
Liquid is at the uniform velocity distributed in outer aqueous phase by SPG films；
S33：When outer aqueous phase engenders milky turbidity emulsion after no longer deepening, double emulsion is transferred into magnetic force stirs
Mix in device, stirring makes dichloromethane volatilize, stir speed (S.S.) 90rpm/min, and whipping temp is 38 DEG C, mixing time 8h；
S34：Double emulsion is transferred in centrifuge, precipitation is collected by centrifugation under the conditions of 4 DEG C, 2700g, distillation water washing is more
After secondary, vacuum freeze drying, water and dichloromethane is distilled completely, it is micro- to obtain load enzyme nanometer described in dry white powder
Bubble.
The present invention is not limited to the above-described embodiments, for those skilled in the art, is not departing from
On the premise of the principle of the invention, some improvements and modifications can also be made, these improvements and modifications are also considered as the protection of the present invention
Within the scope of.The content not being described in detail in this specification belongs to prior art known to professional and technical personnel in the field.
Claims (9)
1. a kind of load enzyme nanometer microvesicle of folacin receptor targeting, the load enzyme nanometer microvesicle are made up of kernel templates and shell, its
It is characterised by：
The kernel templates are CdSe/ZnS quantum dots-compound of coelenteron luciferase 8；
The shell is folic acid cross-linked polyethylene glycol polylactic-co-glycolic acid polymer.
A kind of 2. load enzyme nanometer microvesicle of folacin receptor targeting as claimed in claim 1, it is characterised in that：The CdSe/ZnS
Quantum dot-compound of coelenteron luciferase 8 is formed by CdSe/ZnS quantum dots and the covalent coupling of enteric cavity luciferase 8.
A kind of 3. preparation method of the load enzyme nanometer microvesicle of folacin receptor targeting as claimed in claim 1, it is characterised in that its
Comprise the following steps：
S1：The folic acid cross-linked polyethylene glycol polylactic-co-glycolic acid polymer is prepared, and forms it into the shell；
S2：The CdSe/ZnS quantum dots-compound of coelenteron luciferase 8 is prepared, and forms it into the kernel templates；
S3：The kernel templates are loaded into the shell, complete the preparation for carrying enzyme nanometer microvesicle.
4. the preparation method of the load enzyme nanometer microvesicle of folacin receptor targeting as claimed in claim 3, it is characterised in that institute in S2
It is as follows to state kernel templates preparation method：
1~3ml cushioning liquid is added in glutaraldehyde, and adds 8~12 μ l CdSe/ZnS quantum dots, in ar gas environment
After stirring 1.5~2.5h, separating-purifying simultaneously removes glutaraldehyde；1~3mg coelenterons luciferase 8 is added, is continued in ar gas environment
After 9~11h of stirring reaction, separating-purifying removes coelenteron luciferase 8, obtains the CdSe/ZnS quantum dots-coelenteron fluorescein
The compound of enzyme 8.
5. the preparation method of the load enzyme nanometer microvesicle of folacin receptor targeting as claimed in claim 4, it is characterised in that：It is described slow
It is borate buffer solution or citrate buffer solution to rush solution.
6. the preparation method of the load enzyme nanometer microvesicle of folacin receptor targeting as claimed in claim 4, it is characterised in that：It is described slow
The pH value for rushing solution is 7.2~7.6.
7. the preparation method of the load enzyme nanometer microvesicle of folacin receptor targeting as claimed in claim 3, it is characterised in that will in S3
The preparation method that the kernel templates are loaded into the shell is as follows：
S31：The kernel templates and water are mixed to form interior aqueous phase, by interior aqueous phase be added to the shell concentration for 0~
In 0.050g/ml dichloromethane, colostric fluid is obtained；
S32：The shell and water are mixed to form outer aqueous phase, colostric fluid is distributed in outer aqueous phase using SPG membrane emulsifiers, obtained
To double emulsion；
S33：Double emulsion is transferred in magnetic stirring apparatus, stirring makes dichloromethane volatilize, and stir speed (S.S.) is 80~120rpm/
Min, whipping temp are 30~50 DEG C, and mixing time is 8~10h；
S34：Double emulsion is transferred in centrifuge, precipitation is collected by centrifugation under the conditions of 3~5 DEG C, 2500~3500g, is dried.
8. the preparation method of the load enzyme nanometer microvesicle of folacin receptor targeting as claimed in claim 7, it is characterised in that：Interior aqueous phase
At the uniform velocity it is added to using syringe in dichloromethane.
9. the preparation method of the load enzyme nanometer microvesicle of folacin receptor targeting as claimed in claim 7, it is characterised in that：SPG films
Pressure is 45~55kPa in emulsifier pressure chamber, and colostric fluid is at the uniform velocity distributed in outer aqueous phase by SPG films.
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