CN108904801A - A kind of multifunctional nano vesica and preparation method thereof - Google Patents
A kind of multifunctional nano vesica and preparation method thereof Download PDFInfo
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- CN108904801A CN108904801A CN201810801226.XA CN201810801226A CN108904801A CN 108904801 A CN108904801 A CN 108904801A CN 201810801226 A CN201810801226 A CN 201810801226A CN 108904801 A CN108904801 A CN 108904801A
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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- A61K49/0013—Luminescence
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- A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
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- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
- A61K49/0076—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion
- A61K49/0084—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion liposome, i.e. bilayered vesicular structure
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Abstract
The invention belongs to field of nanometer material technology, and it discloses a kind of multifunctional nano vesicas, including stratum nucleare, shell and the hydrated sheath being arranged in outside shell；Stratum nucleare, shell and the hydrated sheath assembles to form nano vesicle by way of Electrostatic Absorption；The stratum nucleare be in-four (4- carboxyl phenyl) porphines；The shell is polyethyleneimine, and the hydrated sheath is polyethylene glycol hydrated sheath；The shell is modified with the functional group for fluorescing after specifically binding with specific object, or, the stratum nucleare is chelated with magnetic resonance radiography ion or Positron emission computed tomography imaging ion.The purpose of the present invention is to provide a kind of multifunctional nano vesica and its synthetic method, this method synthesizes that bio-toxicity is small, nanoparticles of functional diversities by simple preparation method.
Description
Technical field
The present invention relates to the technical field of nano material, especially a kind of multifunctional nano vesica and its synthetic method.
Background technique
By diagnosing and treating the two clinically independent process integration on a nanoparticle, realization accurately examine immediately
It can synchronize and be treated again while the disconnected state of an illness, be the diagnosis and treatment integrated technique being widely noticed in recent years.Diagnosis and treatment integration nanometer body
While tying up to the instant Precise Diagnosis of realization and synchronous therapeutic, additionally it is possible to by monitoring curative effect to adjust dosage regimen in time,
Reach optimal therapeutic effect, and reduces toxic side effect.Currently, clinically there are a variety of non-intervention type imaging modes, however,
Since the image-forming principle of various imaging techniques is different from method, diagnostic value is also different with limit, this will be generated by list
The insurmountable diagnosis problem of one imaging pattern.Two or more imaging patterns are effectively combined, make it that both there is high score
Resolution, high sensitivity also have preferable tissue penetration, are the directions of medical imaging technology future development.
Fluorescence imaging has many advantages, such as that high sensitivity, the resolution ratio of Single-cell imaging and subcellular rank are a kind of effective
Diagnosing tumor mode.Current main image probe is small organic molecule dyestuff and fluorescent nano material, but organic small point
The disadvantages of sub- dyestuff there are stability poor, poorly water-soluble, photo-labile and acid labile, additionally has certain life
Object toxicity.But the appearance of nano-carrier, it can effectively solve these problems.In addition, nano-material surface is easily modified, it can be simultaneously
Several functional molecules are contained, while improving the biocompatibility of material, are a kind of ideal carriers.
Magnetic resonance imaging mode has high spatial resolution and deep tissue penetration, but its sensitivity is relatively low,
It cannot achieve the other imaging of cell grade.Therefore, in order to improve the exactness and accuracy of clinical diagnosis, magnetic resonance imaging be with it is glimmering
The best affiliate that light imaging building bimodal imaging combines.Magnetic resonance imaging contrast mainly includes two classes at present, a kind of
It is that can promote the longitudinal (T of magnetic resonance containing gadolinium (Gd) complex or derivatives thereof1) imaging effect enhancing, as T1Positive contrast
Agent；Another kind of is superparamagnetic material, can promote magnetic resonance laterally (T2) imaging effect decrease, as T2Negative contrast medium.Cause
This, directly chelates together and is prepared into nanoparticle for fluorescence imaging small molecule and magnetic resonance contrast agent by coordinate bond,
It is a kind of very simple and effective to prepare fluorescence/magnetic resonance bimodal image probe method.
With the continuous development of nano material and nanotechnology, many noninvasive effective technology of cancer treatment are come into being,
Wherein optical dynamic therapy is considered as a kind of efficient, safe, noninvasive and accurate treatment technology.Photosensitizer is as optical dynamic therapy
Key factor, be a kind of chemical substance that luminous energy can be transferred to oxygen molecule around, generate active oxygen radical, such as
Singlet oxygen (1O2), oxygen radical can irreversibly destroy membrane structure, cause pathological tissues cell dead.
Key factor of the photosensitizer as optical dynamic therapy selects most important.Currently used photosensitizer mainly wraps
Small organic molecule dyestuff and inorganic nanoparticles are included, inorganic nanoparticles are mainly up-conversion nanoparticles, rare earth quantum dot etc..
Up-conversion nanoparticles and rare earth quantum dot stability are high, have can across ultra-violet (UV) band and the wider absorption spectra of visible region, but
It is the rare earth element that its main raw material(s) is inorganic matter element, bio-toxicity and metabolic problems seriously limit its application.
However, the main component of small organic molecule dyestuff is carbon, nitrogen, hydrogen and oxygen etc., and it is consistent with organism component, have more
For wide application prospect.
In conclusion above-mentioned technology has problems in that：It is not available simple and effective means and constructs biological poison
Property small, preparation simplify, the nano material of the optical dynamic therapy of the multi-modality imagings of functional diversities guidance.
Summary of the invention
The present invention is intended to provide a kind of multifunctional nano vesica and its synthetic method, this method pass through simple preparation method
Synthesize that bio-toxicity is small, nanoparticles of functional diversities.
Before illustrating the solution of the present invention, make an explanation to following English abbreviation：FL：Fluorescence；MR：Magnetic resonance；
PETCT：Positron emission computed tomography；PDT：Optical dynamic therapy；TCPP：In-four (4- carboxyl phenyl) porphines；EDC：
1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride；NHS:N- hydroxysuccinimide；mPEG-NH2:Amino is repaired
The polyethylene glycol of decorations；γ-PGA：Polyglutamic acid；PEI：Polyethyleneimine.
Its concrete scheme is：A kind of multifunctional nano vesica, including stratum nucleare, shell and the hydrated sheath being arranged in outside shell；
Stratum nucleare, shell and the hydrated sheath assembles to form nano vesicle by way of Electrostatic Absorption；
The stratum nucleare be in-four (4- carboxyl phenyl) porphines；The shell is polyethyleneimine, the hydrated sheath
For polyethylene glycol hydrated sheath；
The shell is modified with the functional group for fluorescing after specifically binding with specific object, or, institute
The stratum nucleare stated is chelated with magnetic resonance radiography ion or Positron emission computed tomography imaging ion.
In the upper multifunctional nano vesica, the functional group includes but is not limited to zinc ion recognition group
Or copper ion recognition group, the functional group and shell are by being covalently keyed.
Meanwhile the invention also discloses a kind of preparation method of multifunctional nano vesica as described above ,-four (4- by
Carboxyl phenyl) porphines, polyethyleneimine, polyglutamic acid modification polyethylene glycol three is mixed at room temperature for a period of time be
It can；
(4- carboxyl phenyl) porphines is chelated with magnetic resonance radiography ion or positron emission tomography-four in described
The branched modified of scanning imagery ion or the polyethyleneimine has for sending out glimmering after specifically binding with specific object
The functional group of light.
In the preparation method of above-mentioned multifunctional nano vesica, the mixing time is 5-24 hours.
In the preparation method of above-mentioned multifunctional nano vesica, the functional group is zinc ion recognition group or copper
Ion identification group, the functional group and shell are by being covalently keyed.
In the preparation method of above-mentioned multifunctional nano vesica, the magnetic resonance radiography ion or positron emission meter
Calculation machine Tomography ion is Gd3+、64Cu or Mn2+。
It further include dialysis step in the preparation method of above-mentioned multifunctional nano vesica：Being mixed, end is laggard
Row dialysis purification.
The beneficial effects of the present invention are：
Polyethyleneimine molecular weight of the present invention is smaller, molecular weight ranges 8000-12000, and molecular toxicity is small.
More importantly synthetic method of the present invention is mildly simple, the covalent linkage of polyethyleneimine surface not will lead to
Functional group fracture.It is conducive to least functional group and with maximum likelihood be connected to hydrophilic polyethylene as far as possible
The surface of imines.
The effect of in order to further increase polyethyleneimine surface ion recognition group of the invention, method of the invention are closed
It is ball-type nano particle at obtained particle, the ion identification group of nano grain surface can be as straight with effects of ion as possible
Contact, and surface can be high, and detection efficiency is caused to significantly improve.
Synthetic method of the invention is simply mild, and it is difficult to overcome the big technology of synthesis difficulty generally existing in traditional technology
Point.
Another advantage thereof is that：TCPP itself has fluorescent effect, when PEI surface modification be used for it is specific
Object specific binding after after the functional group that fluoresces, then the fluorescence of TCPP can be used as internal standard, due to functional group
Another fluorescence color is presented, by the fluorescence of functional group and the ratio of internal standard fluorescence, it is negative that photobleaching, probe can be eliminated
Data distortion caused by load and retention and apparatus factor (excitation photostability), can effectively improve detection accuracy, especially carefully
Detection intracellular.
Detailed description of the invention
Fig. 1 is the transmission electron microscope photo of the embodiment of the present invention 1；
Fig. 2 is the fluorescence excitation and emission spectra figure of the embodiment of the present invention 1；
Fig. 3 is the various concentration solution T of the embodiment of the present invention 11Signal strength map；
Fig. 4 is the various concentration solution and T of the embodiment of the present invention 11 -1The correlogram of value；
Fig. 5 is the cytotoxicity test of the various concentration solution of the embodiment of the present invention 1；
Fig. 6 is the hemolytic test of the various concentration solution of the embodiment of the present invention 1；
Fig. 7 is of the invention using the self assembling process of the multifunctional nano vesica based on electrostatic self-assembled and its two kinds
Different purposes.
Specific embodiment
With reference to embodiment, technical solution of the present invention is described in further detail, but do not constituted pair
Any restrictions of the invention.
In order to which more clearly the present invention will be described, embodiment is listed below to illustrate superiority of the invention.
Embodiment 1
Optical dynamic therapy (PDT) nano vesicle (Gd-PNPs) under fluorescence (FL)/magnetic resonance (MR) bimodal imaging guidance
Preparation：
A, the preparation of Gd-TCPP：It is anhydrous that 6g imidazoles, 8mg TCPP and excess are added in the three-neck flask of a 250mL
GdCl3(27mg).220 DEG C at a temperature of and N2It is stirred to react 2h under environment, then product is dissolved in the methanol of 10mL,
It is kept in dark place in 4 DEG C of refrigerators spare.During the reaction, imidazoles gradually recrystallizes precipitation in bottleneck；
B, the preparation of mPEG-PGA：γ-PGA, mPEG-NH2, EDC and NHS are dissolved in 5mL phosphate buffer PBS (pH
=8.5) in, wherein molar ratio is 1:3.5:3.85:3.85, mixture is stirred at room temperature for 24 hours.Product is by deionized water dialysis
It is then freeze-dried after purifying 2 days, is placed in drier and saves backup；
C, the Gd-TCCP solution 0.3mL and 5mL PEI aqueous solution (0.5mgmL of above-mentioned synthesis are taken-1) mixing, stirring
Then 1mL mPEG-PGA (1mgmL is added in 12h-1), mixed solution is stirred into 1h.Final purification deionized water dialysis
For 24 hours, Gd-PNPs after purification is obtained.
TCPP：In-four (4- carboxyl phenyl) porphines；EDC：1- (3- dimethylamino-propyl) -3- ethyl carbodiimide hydrochloride
Salt；NHS:N- hydroxysuccinimide；mPEG-NH2:Amido modified polyethylene glycol；γ-PGA：Polyglutamic acid；PEI：Poly- second
Alkene imines.
EDC, NHS activated carboxyl together promote the amino on carboxyl and mPEG-NH2 on PGA to react.
In reaction process is added, Gd3+It can be sequestered in the tetrapyrrol(e) structure of TCPP by coordinate bond, it is anti-in self assembly
During answering, since, containing a large amount of positively charged amino, hydrophobic Gd-TCPP contains electronegative in hydrophilic PEI
Carboxyl acts on forming nano vesicle by Electrostatic Absorption and hydrophobe；In the prior art, there is individual case in organic solvent
In, TCPP and PEI is subjected to the assembling based on amidation process, this package technique scheme is complicated, and for the function of grafting
Group is affected.
In the present embodiment, hydrophilic PEI is assembled in the surface of nano vesicle, and hydrophobic Gd-TCPP, which is assembled in, to be received
Inside rice vesica.Gd3+Presence may make the nanoparticle of preparation that there is the longitudinal (T of magnetic resonance1) contrast ability, while TCPP can
As quick dose of fluorescence imaging molecule and PDT light, therefore there is prepared nano vesicle fluorescence/magnetic resonance bimodal function is imaged
Energy and PDT effect, are a kind of integrated multifunctional nano vesicas of diagnosis and treatment.
Transmission electron microscope result
It can be seen that Gd-PNPs partial size prepared by embodiment 1 is about 180nm in transmission electron microscope image (Fig. 1),
And even particle size, it is well dispersed.
Fluorescence property result
It is observed that its maximum emission peak preferably resists in 660nm in fluorescence excitation and emission spectra figure (Fig. 2)
Biological context fluorescence interference performance and tissue penetration have the present invention and draw applied to clinically fluorescence localization lesions position
Lead the potentiality and prospect of operation and immunofluorescence pathological analysis.
Relaxation rate result
The T of various concentration gradient G d-PNPs solution1(Fig. 3, wherein abscissa is Gd to figure3+Concentration, when ordinate is relaxation
Between inverse) display embodiment 1 prepared by Gd-PNPs have signal strength with concentration increase and enhance property.By not
With concentration Gd-PNPs and T1 -1Its available T of correlogram (Fig. 4)1Relaxation rate is 16.157mM-1·s-1。
The following table 1 lists the relaxation rate of Gd-PNPs prepared by embodiment 1 and three kinds of commercial comparison's agent.
Relaxation rate (the unit of 1 four kinds of contrast medium of table：mM-1·s-1)
The relaxation rate of contrast medium containing gadolinium that this new polymers nano particle is shown by the comparing result of upper table is three kinds of quotient
4 times of product contrast medium relaxation rate may infer that the novel contrast medium dosage can drop under conditions of reaching identical reduction of contrast signal
Down to a quarter, contrast medium dosage reduces the incidence that can reduce contrast induced nephropathy, therefore the development of this novel contrast medium
With practical application value.
Biocompatibility result
(Fig. 5, wherein abscissa is nano vesicle concentration to the cytotoxicity test of various concentration Gd-PNPs, and ordinate is thin
Born of the same parents' activity) in it can be seen that at concentrations up to about 400 μ gmL-1The survival rate of lower cell, therefore can be with still 90% or more
Conclude that its cytotoxicity is lower.At the same time, (Fig. 6, wherein abscissa is nanometer for the hemolytic test of various concentration Gd-FPNPs
Vesica concentration, ordinate be cell hemolysis rate) in it can also be seen that at concentrations up to 400 μ gmL-1Under conditions of, in solution
Erythrocyte still do not destroy significantly, it was demonstrated that its hemolytic is lower, have good blood compatibility.
By significantly reducing material in the preferable polyethylene glycol of nano vesicle surface modification biocompatibility (PEG)
Bio-toxicity improves its biocompatibility.
The present invention also pass through in-four (4- carboxyl phenyl) porphines (TCPP) and Gd3+Coordinate bond by Gd3+It is sequestered in TCPP
On, so that it is provided simultaneously with fluorescence imaging and magnetic resonance imaging function.
Specifically, since gadolinium contains 7 non-coordinated electronics, coordinate bond can be generated with the tetrapyrrole ring in TCPP structure
It is joined together to form metalloporphyrin chelate.TCPP is in addition to being a kind of fluorescence imaging probe, and a kind of good smooth power
The photosensitizer for the treatment of chelates gadolinium and TCPP together, its function can be made to enhance, and has fluorescence/magnetic resonance bimodal imaging
Ability.The form of coordinate bond simultaneously, but also Gd3+It is not likely to produce leakage.
The present invention by above-mentioned improvement so that prepared nano vesicle not only have fluorescence/magnetic resonance bimodal at
As ability, also there is optical dynamic therapy effect.Meanwhile the form of nano vesicle, so that it is with good nano material property.
The modification of polyethylene glycol, so that its biocompatibility greatly improves.In conclusion we be prepared a kind of collection fluorescence imaging,
Magnetic resonance imaging, optical dynamic therapy are in the multifunctional nano vesica of one.
Embodiment 2
Optical dynamic therapy under fluorescence (FL)/Positron emission computed tomography (PETCT) bimodal imaging guidance
(PDT) preparation of nano vesicle (Gd-PNPs)：
The preparation of A.mPEG-PGA：γ-PGA, mPEG-NH2, EDC and NHS are dissolved in 5mL phosphate buffer PBS (pH
=8.5) in, wherein molar ratio is 1:3.5:3.85:3.85, mixture is stirred at room temperature for 24 hours.Product is by deionized water dialysis
It is then freeze-dried after purifying 2 days, is placed in drier and saves backup.
B. the methanol solution 0.3mL (0.8mgmL of TCCP is taken-1) and 5mL PEI aqueous solution (0.5mgmL-1) mixing,
5h is stirred, is then sequentially added a certain amount of64Cu2+Solution, 1mL mPEG-PGA (1mgmL-1), mixed solution is stirred into 1h.
Final purification for 24 hours, is obtained after purification with deionized water dialysis64Cu-PNPs。
TCPP：In-four (4- carboxyl phenyl) porphines；EDC：1- (3- dimethylamino-propyl) -3- ethyl carbodiimide hydrochloride
Salt；NHS:N- hydroxysuccinimide；mPEG-NH2:Amido modified polyethylene glycol；γ-PGA：Polyglutamic acid；PEI：Poly- second
Alkene imines.
It is hydrophobic due to containing a large amount of positively charged amino in hydrophilic PEI during self-assembling reaction
TCPP contains electronegative carboxyl, acts on forming nano vesicle by Electrostatic Absorption and hydrophobe, and hydrophilic PEI is assembled in
The surface of nano vesicle, hydrophobic TCPP are assembled in inside nano vesicle.In addition,64Cu2+It can be sequestered in by coordinate bond
In the tetrapyrrol(e) structure of TCPP,64Cu2+Presence may make the nanoparticle of preparation that there is PETCT contrast ability, while TCPP
It can be used as fluorescence imaging molecule and quick dose of PDT light, therefore prepared nano vesicle has fluorescence/positron emission emission computer
Tomoscan bimodal imaging function and PDT effect are a kind of integrated multifunctional nano vesicas of diagnosis and treatment.
Embodiment 3
Optical dynamic therapy (PDT) nano vesicle (Mn-PNPs) under fluorescence (FL)/magnetic resonance (MR) bimodal imaging guidance
Preparation：
The preparation of A.mPEG-PGA：γ-PGA, mPEG-NH2, EDC and NHS are dissolved in 5mL phosphate buffer PBS (pH
=8.5) in, wherein molar ratio is 1:3.5:3.85:3.85, mixture is stirred at room temperature for 24 hours.Product is by deionized water dialysis
It is then freeze-dried after purifying 2 days, is placed in drier and saves backup；
B. the methanol solution 0.3mL (0.8mgmL of TCCP is taken-1) and 5mL PEI aqueous solution (0.5mgmL-1) mixing,
Stirring for 24 hours, then sequentially adds a certain amount of Mn2+Solution, 1mL mPEG-PGA (1mgmL-1), mixed solution is stirred into 1h.
Final purification for 24 hours, obtains Mn-PNPs after purification with deionized water dialysis.
TCPP：In-four (4- carboxyl phenyl) porphines；EDC：1- (3- dimethylamino-propyl) -3- ethyl carbodiimide hydrochloride
Salt；NHS:N- hydroxysuccinimide；mPEG-NH2:Amido modified polyethylene glycol；γ-PGA：Polyglutamic acid；PEI：Poly- second
Alkene imines.
It is hydrophobic due to containing a large amount of positively charged amino in hydrophilic PEI during self-assembling reaction
TCPP contains electronegative carboxyl, acts on forming nano vesicle by Electrostatic Absorption and hydrophobe, and hydrophilic PEI is assembled in
The surface of nano vesicle, hydrophobic TCPP are assembled in inside nano vesicle.In addition, Mn2+It can be sequestered in by coordinate bond
In the tetrapyrrol(e) structure of TCPP, Mn2+It can be used as magnetic resonance transverse direction image-forming contrast medium, existing may make the nanoparticle of preparation to have
There is mr angiography ability, while TCPP can be used as fluorescence imaging molecule and quick dose of PDT light, therefore prepared nano vesicle has
Fluorescence/magnetic resonance bimodal imaging function and PDT effect are a kind of integrated multifunctional nano vesicas of diagnosis and treatment.
Embodiment 4
Zinc ion (Zn2+)/pH ratio fluorescent detects the preparation of nano-probe (Q-PNPs)：
A.8- the preparation of (2- chloroacetamide) quinoline (Q)：First by 2.88g 8- aminoquinoline, 3mL triethylamine, 10mL dichloro
Methane is added in 50mL three-neck flask, then flask is placed in ice bath, then, 5.31mL chloracetyl chloride and 5mL bis- is slowly added dropwise
The mixed liquor of chloromethanes reacts 2h.Then, using the pH of manganese hydrogen sodium regulating solution to 7.It is direct with funnel after having adjusted pH
Filtering collects filtrate, filtrate is then evaporated under reduced pressure, the resulting a small amount of methylene chloride of brownish black solid is dissolved, is then used
Column chromatography isolates 8- (2- chloroacetamide) quinoline solution.Finally, being evaporated under reduced pressure, 8- (2- chloroacetamide) quinoline can be obtained
Solid；
The preparation of B.PEIQ：By 0.5g PEI, 0.2g 8- (2- chloroacetamide) quinoline, 0.092g KI, 0.14g K2CO3
It is dissolved in 20mL acetonitrile, and the heated overnight at reflux in 80 DEG C of oil baths, to obtain PEIQ crude product, then passes through reduction vaporization
Solvent is co-precipitated 3 times with ether, finally uses pure water dialysis purification；
The preparation of C.mPEG-PGA：γ-PGA, mPEG-NH2, EDC and NHS are dissolved in 5mL phosphate buffer PBS (pH
=8.5) in, wherein molar ratio is 1:3.5:3.85:3.85, mixture is stirred at room temperature for 24 hours.Product is by deionized water dialysis
It is then freeze-dried after purifying 2 days, is placed in drier and saves backup；
D. the methanol solution 0.3mL (0.8mgmL of TCCP is taken-1) and 5mL PEIQ aqueous solution (0.8mgmL-1) mixing,
For 24 hours, 1mL mPEG-PGA (1mgmL is then added in stirring-1), mixed solution is stirred into 1h.Final purification deionized water is saturating
Analysis for 24 hours, obtains Q-PNPs after purification.
TCPP：In-four (4- carboxyl phenyl) porphines；EDC：1- (3- dimethylamino-propyl) -3- ethyl carbodiimide hydrochloride
Salt；NHS:N- hydroxysuccinimide；mPEG-NH2:Amido modified polyethylene glycol；γ-PGA：Polyglutamic acid；PEI：Poly- second
Alkene imines.
It is hydrophobic due to containing a large amount of positively charged amino in hydrophilic PEIQ during self-assembling reaction
TCPP contains electronegative carboxyl, acts on forming nano vesicle by Electrostatic Absorption and hydrophobe, hydrophilic PEIQ assembling
On the surface of nano vesicle, hydrophobic TCPP is assembled in inside nano vesicle.In addition, Q can specificity and Zn2+In conjunction with,
Green fluorescence can be generated, and with Zn2+Content increases, and fluorescence intensity enhancing can be used as Zn2+Detection probe, in addition PEI
Also it can be used as water proton detection probe, prepared Q-PNPs has response performance to pH, can be used as pH detection probe.Therefore institute
The nano vesicle of preparation can detect Zn2+And pH, it is a double base detection nano-probe.
In addition, in the present embodiment, TCPP itself is displayed in red fluorescence, and 8- (2- chloroacetamide) quinoline and Zn2+Specifically
Property combine after can show green fluorescence.In the detection process, by two different colors of fluorescence ratio, red fluorescence can be made
For internal standard, in the situation known to TCPP concentration, red fluorescence can be set to standard 1, then pass through different colours fluorescence
Intensity rate, Zn can accurately be calculated2+Concentration, need not move through other steps and calibrated and corrected, accuracy
It is high.
Embodiment 5
Copper ion (Zn2+)/pH ratio fluorescent detects the preparation of nano-probe (R-PNPs)：
The preparation of A.PEIR：0.5g PEI, 0.6g rhodamine 6G (R) are dissolved in 20mL ethyl alcohol, and in 80 DEG C of oil
Heated overnight at reflux in bath passes through pure water dialysis purification to obtain PEIR crude product；
The preparation of B.mPEG-PGA：γ-PGA, mPEG-NH2, EDC and NHS are dissolved in 5mL phosphate buffer PBS (pH
=8.5) in, wherein molar ratio is 1:3.5:3.85:3.85, mixture is stirred at room temperature for 24 hours.Product is by deionized water dialysis
It is then freeze-dried after purifying 2 days, is placed in drier and saves backup；
C. take methanol solution 0.3mL (0.8mgmL-1) and the 5mL PEIR aqueous solution (0.8mgmL-1) of TCCP mixed
It closes, stirs 16h, 1mL mPEG-PGA (1mgmL-1) then is added, mixed solution is stirred into 1h.Final purification deionization
Water is dialysed for 24 hours, and R-PNPs after purification is obtained.
TCPP：In-four (4- carboxyl phenyl) porphines；EDC：1- (3- dimethylamino-propyl) -3- ethyl carbodiimide hydrochloride
Salt；NHS:N- hydroxysuccinimide；mPEG-NH2:Amido modified polyethylene glycol；γ-PGA：Polyglutamic acid；PEI：Poly- second
Alkene imines.
It is hydrophobic due to containing a large amount of positively charged amino in hydrophilic PEIR during self-assembling reaction
TCPP contains electronegative carboxyl, acts on forming nano vesicle by Electrostatic Absorption and hydrophobe, hydrophilic PEIR assembling
On the surface of nano vesicle, hydrophobic TCPP is assembled in inside nano vesicle.In addition, R can specificity and Cu2+In conjunction with,
Blue-fluorescence can be generated, and with Cu2+Content increases, and fluorescence intensity enhancing can be used as Cu2+Detection probe, in addition PEI
Also it can be used as water proton detection probe, prepared R-PNPs has response performance to pH, can be used as pH detection probe.Therefore institute
The nano vesicle of preparation can detect Cu2+And pH, it is a double base detection nano-probe.
Fig. 7 shows the self assembling process and its two kinds of differences using the multifunctional nano vesica based on electrostatic self-assembled
Purposes.
Above-described is only presently preferred embodiments of the present invention, all made within the scope of the spirit and principles in the present invention
What modifications, equivalent substitutions and improvements etc., should all be included in the protection scope of the present invention.
Claims (7)
1. a kind of multifunctional nano vesica, which is characterized in that including stratum nucleare, shell and the hydrated sheath being arranged in outside shell；It is described
Stratum nucleare, shell and hydrated sheath assemble to form nano vesicle by way of Electrostatic Absorption；
The stratum nucleare be in-four (4- carboxyl phenyl) porphines；The shell is polyethyleneimine, and the hydrated sheath is poly-
Ethylene glycol hydrated sheath；
The shell is modified with the functional group for fluorescing after specifically binding with specific object, or, described
Stratum nucleare is chelated with magnetic resonance radiography ion or Positron emission computed tomography imaging ion.
2. multifunctional nano vesica according to claim 1, which is characterized in that the functional group is to include but not office
It is limited to zinc ion recognition group or copper ion recognition group, the functional group and shell are by being covalently keyed.
3. a kind of preparation method of multifunctional nano vesica as described in claim 1, which is characterized in that-four (the 4- carboxyls by
Phenyl) porphines, polyethyleneimine, polyglutamic acid modification polyethylene glycol three a period of time is mixed at room temperature；
(4- carboxyl phenyl) porphines is chelated with magnetic resonance radiography ion or Positron emission computed tomography-four in described
The branched modified of imaging ion or the polyethyleneimine have for fluoresce after the specific binding of specific object
Functional group.
4. the preparation method of multifunctional nano vesica according to claim 3, which is characterized in that the incorporation time is
5-24 hours.
5. the preparation method of multifunctional nano vesica according to claim 3, which is characterized in that the functional group packet
Zinc ion recognition group or copper ion recognition group are included but are not limited to, the functional group and shell are connected by covalent bond
It connects.
6. the preparation method of multifunctional nano vesica according to claim 3, which is characterized in that the magnetic resonance radiography
Ion or Positron emission computed tomography imaging ion are Gd3+、64Cu or Mn2+。
7. the preparation method of multifunctional nano vesica according to claim 3, which is characterized in that further include dialysis step：
Dialysis purification is carried out after mixing.
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| CN114073922A (en) * | 2020-08-20 | 2022-02-22 | 中国科学院苏州纳米技术与纳米仿生研究所 | Porphyrin-based metal-organic framework nanosphere and preparation method and application thereof |
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| CN114073922A (en) * | 2020-08-20 | 2022-02-22 | 中国科学院苏州纳米技术与纳米仿生研究所 | Porphyrin-based metal-organic framework nanosphere and preparation method and application thereof |
| CN114073922B (en) * | 2020-08-20 | 2024-02-20 | 中国科学院苏州纳米技术与纳米仿生研究所 | Porphyrin-based metal-organic framework nanosphere and preparation method and application thereof |
| CN116496786A (en) * | 2023-07-01 | 2023-07-28 | 北京建工环境修复股份有限公司 | Gadolinium nanocluster fluorescent probe and application thereof in environment detection |
| CN116496786B (en) * | 2023-07-01 | 2023-09-29 | 北京建工环境修复股份有限公司 | Gadolinium nanocluster fluorescent probe and application thereof in environment detection |
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