CN107496940B - Height carries the melanin sample nano material and preparation method and application of manganese amount and high relaxation rate - Google Patents
Height carries the melanin sample nano material and preparation method and application of manganese amount and high relaxation rate Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
- A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
- A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
- A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
- A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
- A61K49/1851—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule
- A61K49/1857—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule the organic macromolecular compound being obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. PLGA
- A61K49/186—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule the organic macromolecular compound being obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. PLGA the organic macromolecular compound being polyethyleneglycol [PEG]
Abstract
The present invention relates to a kind of high melanin sample nano materials and preparation method and application for carrying manganese amount and high relaxation rate, the nano material is using potassium permanganate as manganese source and oxidant, it is polymerized as precursor molecule by levodopa, is the melanin sample nano particle MnEMNPs of additive Mn.The material is the melanin sample nano particle for having superior water dispersibility, have it is high carry manganese amount (>10%) with geometry restriction effect, excellent T is shown1‑T2Double mode MRI Contrast enhanced abilities.Preparation method of the present invention is easy, mild, environmentally protective, and new approaches and new way are provided for exploitation high-efficiency MRI contrast agent.
Description
Technical field
The invention belongs to the technical fields of functionalized macromolecular polymer, and in particular to a kind of high load manganese amount and high relaxation rate
Melanin sample nano material and preparation method and application.
Background technology
Magnetic resonance imaging (MRI) is a kind of valuable clinical imaging technique.In order to improve specificity, sensibility
And diagnostic accuracy, it is developed there are many MRI nanometers of contrast medium at present, including positivity contrast medium (T1) and negativity contrast medium (T2)。
Recently, T1-T2Double mode MRI contrast agent receives very big concern, and compared with single-mode MRI contrast agent, it is capable of providing comprehensively
Collaborative diagnosis information, improve diagnostic accuracy.Geometry limitation can freely overturning and adjacent to moisture by limitation comparison agent molecule
The diffusion of son improves contrast medium spin correlation time and water diffusion correlation time, to improve the relaxation rate of contrast medium, quilt
It is considered to prepare the available strategy of high-performance MRI contrast agent.
With simply by T1Contrast material and T2Contrast material mixes difference, by the T of high concentration1Component is entrained in
It, can be simultaneously in non magnetic meso-porous matrix (such as polymer, albumin nanoparticle, mesoporous silicon, metal organic frame, hydrogel)
Obtain T1-T2Double mode MRI Contrast enhanced effects.In this case, the geometry restriction effect caused by unique texture, week
The coordination and Chemical Exchange for enclosing hydrone are restricted, and lead to r1Relaxation rate improves.Meanwhile concentration effect can influence T2Relaxation
Average effect is moved, r is improved2Relaxation rate.
Constituent inorganic matter of Conventional nano material itself is had, in vivo prolonged stay, it is difficult to it is metabolized,
There are safety issues.Melanin is a kind of natural polymer with excellent metal ion chelating capacity, is widely present in life
Object respectively in tissue, has good biocompatibility.At present there are many paramagnetic metal ion (such as manganese ion, iron ion,
Gadolinium ion etc.) it is sequestered in melanin sample nano grain surface, as MRI contrast agent.However, the document reported at present is all first
First (such as sodium hydroxide, ammonium hydroxide) passes through chemistry or the suitable precursor molecule of enzymatic oxidation in harmful strong alkali environment
(such as Dopamine hydrochloride, tyrosine, levodopa) obtains melanin sample nano particle, metal ion is then added, by it
It is sequestered in the melanin sample nano grain surface synthesized, that is, strategy is adulterated after polymerizeing.This strategy has following defect：(1)
Nano particle synthesis, chelating and purification step are comparatively laborious；(2) metal ion delivered payload capability is very low (usual ＜ 1%), causes
MRI relaxation rates are relatively low；(3) metal ion chelated has the risk to fall off from particle surface；(4) water dispersible is bad.Therefore,
It urgently develops easy synthetic method and prepares the high load manganese amount melanin sample nano particle with superior water dispersibility, this is for carrying
Its high biologic applications and MRI relaxation rates have important scientific meaning.
Invention content
In view of the deficiencies of the prior art, the present invention provides a kind of high melanin sample nanometer materials for carrying manganese amount and high relaxation rate
Material, the nano material is melanin nano particle (the manganese doped of the additive Mn with superior water dispersibility
Eumelanin nanocomposites, MnEMNPs), have it is high carry manganese amount (>10%) it with geometry restriction effect, shows excellent
Different T1-T2Double mode MRI Contrast enhanced abilities.Preparation method of the present invention is easy, mild, environmentally protective, novel to develop
High-effect MRI contrast agent new approaches and new way are provided.
The technical scheme is that：
Height carries the melanin sample nano material of manganese amount and high relaxation rate, and by manganese source and oxidant, levodopa is as forerunner
Body molecule aggregation forms, and is the melanin sample nano particle MnEMNPs of additive Mn.
Using potassium permanganate as manganese source and oxidant.
Height carries the preparation method of the melanin sample nano material of manganese amount and high relaxation rate, there is following steps：
By levodopa suspension heating water bath to 50 DEG C, liquor potassic permanganate is taken, is rapidly joined under intense agitation
It into levodopa suspension, reacts 6 hours, centrifugation is resuspended for several times, and the MnEMNPs of acquisition is scattered in deionized water.It is prepared
Process is as follows：
The levodopa：The molar ratio of potassium permanganate is 1：0.3.
The liquor potassic permanganate is the liquor potassic permanganate of ultrasonic disperse.
It is 4-5 times that the centrifugation, which is resuspended, and centrifugal speed is 17500 revs/min, is centrifuged 15 minutes.
Height carries melanin sample nano material the answering in preparing for magnetic resonance imaging contrast agent of manganese amount and high relaxation rate
With.
The preparation is T1-T2Double mode MRI contrast agent.
Nano material (particle) of the present invention is using potassium permanganate as oxidant and manganese source, and levodopa is as presoma
Molecule is obtained by adulterating strategy in polymerization.The nano material be it is water-dispersed it is high carry manganese amount (>And high relaxation rate 10%)
(under 1.5T field strength, r1Up to 60.8mM-1s-1) MnEMNPs.Applicant it is demonstrated experimentally that its under 1.5T field strength, longitudinal relaxation
Rate r1Value is 9 times or so of clinical gadolinium agent.The forming process of nano particle is related to oxidation, rearrangement, molecule cyclisation, polymerization and manganese chela
Multiple steps such as conjunction.In the course of the polymerization process, it since melanin molecules surface has abundant metal ion chelating site, is reduced
It can continuously be doped into nano particle at the manganese ion of low price.The preparation method is easy, mild, environmentally protective, repeats
Property it is high, since the manganese ion in material of the present invention is a kind of trace element necessary to organism life process, organism
Its controllable stable state.Melanin also is respectively organized to be widely present into the cell in organism, can be effectively metabolized.Therefore, this material is complete
It is made of entirely the natural constituents of organism, avoids the safety issue of Conventional nano material, there is greatly clinical conversion valence
Value.
Description of the drawings
Fig. 1 is for the synthesis of MnEMNPs and its as T1-T2The application schematic diagram of double mode MRI contrast agent；
Fig. 2 is the uv-visible absorption spectroscopy of different time points reaction solution in building-up process；
Fig. 3 is the Physickemical Properties of MnEMNPs；Wherein, Fig. 3 a are transmission electron microscope picture；Fig. 3 b are total for electron spin
Shake spectrogram；Fig. 3 c are Raman spectrum；Fig. 3 d scanning electron microscope (SEM) photographs and elemental analysis (carbon, oxygen, manganese)；Fig. 3 e are Mn2p1/2And Mn2p3/2
X-ray photoelectron spectroscopic analysis；Fig. 3 f are that Inductively coupled plasma mass spectrometry detects manganese stability；
The magnetic performance that Fig. 4 is MnEMNPs characterizes；Wherein, Fig. 4 a are vibrating specimen magnetometer testing result；Fig. 4 b and 4c
For Hydrogen Proton nuclear magnetic resonance dispersion results；Fig. 4 d are the T of MnEMNPs under different field strength1Weighted sum T2Weighted image；Fig. 4 e are not
With the r of MnEMNPs under field strength1Relaxation rate；Fig. 4 f are the r of MnEMNPs under different field strength2Relaxation rate；
Fig. 5 is Mn under different field strength2+The MRI image and r of standard items and Gd-DTPA1Relaxation rate；
Pegylation characterization, colloidal stability and the blood compatibility experiment that Fig. 6 is MnEMNPs；Wherein, Fig. 6 a are to receive
The uv-visible absorption spectroscopy of rice grain；Fig. 6 b are the size of hydration distribution map of nano particle.Fig. 6 c are Fu of nano particle
In leaf transformation infrared spectrum；Fig. 6 d are the UV-Visible absorption light after MnEMNPs is incubated 24 hours in different physiological solutions
Spectrum；Fig. 6 e are the uv-visible absorption spectroscopy after PMnEMNPs is incubated 24 hours in different physiological solutions；Fig. 6 f are nanometer
The blood compatibility test result of particle；Illustration in Fig. 6 f is corresponding picture；
The cellular uptake experiment and cell imaging that Fig. 7 is PMnEMNPs；Wherein, Fig. 7 a are that 50 μ g/mL PMnEMNPs are incubated
U87MG cell optical microscopes after 6 hours.Upper row is control group, and lower row is experimental group.Scale：100 μm (left sides), 50 μm
(right side)；Fig. 7 b are the U87MG cell transmission electron microscope pictures after 50 μ g/mL PMnEMNPs are incubated 6 hours, and upper row is control group.Lower row
For experimental group.Scale：2 μm (left sides), 1 μm (right side)；Fig. 7 c are the U87MG cell activity inspection after various concentration PMnEMNPs is incubated
It surveys；Fig. 7 d are that Inductively coupled plasma mass spectrometry quantitatively detects cellular uptake PMnEMNPs；Fig. 7 e are that MRI detects various concentration
The T of U87MG cells after PMnEMNPs incubations1Relaxation time；Illustration in Fig. 7 e is corresponding cell T1Weighted image；Fig. 7 f
The T of the U87MG cells after various concentration PMnEMNPs is incubated is detected for MRI2Relaxation time；Illustration in Fig. 7 f is corresponding thin
Born of the same parents T2Weighted image；
Fig. 8 is U87MG mice with tumor MRI；Wherein, Fig. 8 a are that U87MG mice with tumor is unenhanced and through tail vein injection PMnEMNPs
The T of different time points afterwards1Weighted image (on) and T2Weighted image (under)；Fig. 8 b be U87MG mice with tumor tumor locus it is unenhanced and note
Penetrate the T of different time points after PMnEMNPs1Relaxation time changes；Fig. 8 c be U87MG mice with tumor tumor locus it is unenhanced and injection
The T of different time points after PMnEMNPs2Relaxation time changes；
Fig. 9 is the serum biochemistry of healthy mice different time points (3 days, 7 days, 14 days) after tail vein injection PMnEMNPs
Testing result；
Figure 10 is healthy mice main organs (brain, the heart, liver, spleen, lung, kidney) after tail vein injection PMnEMNPs is 15 days
Hematoxylin eosin staining figure；
Figure 11 is mouse weight variation diagram of the healthy mice in tail vein injection PMnEMNPs 15 days.
Specific implementation mode
The reagent that the present invention uses：Levodopa, potassium permanganate, manganese ion standard items purchased from Aladdin Reagent Company (on
Sea, China).Magnevist Solution (Gd-DTPA, magnevist) is purchased from Kang Chen medicine companies Group Co., Ltd (Guangzhou, China).
Deionized water is obtained by Mi Libo ultrapure water systems.
The preparation of 1.MnEMNPs：
Referring to Fig. 1, by 60mL 10mM levodopa suspension heating water baths to 50 DEG C.Take the 1.8mL of advance ultrasonic disperse
100mM liquor potassic permanganates rapidly join that (levodopa/potassium permanganate feeds intake to levodopa suspension under intense agitation
Molar ratio is 1：0.3).Reaction carries out 6 hours under intense agitation.Centrifugation be resuspended 4-5 times (17500 revs/min, 15 points
Clock), to remove extra precursors and by-product.Finally, the MnEMNPs of acquisition is scattered in deionized water.
The Physico-Chemical Characterization of 2.MnEMNPs：
Different time points during the reaction take 20 μ L reaction solutions, measure its uv-visible absorption spectroscopy, right
Reaction process carries out dynamic monitoring.It by freeze dryer freeze-drying, weighs, MnEMNPs is quantified.Chloroazotic acid clears up sample, inductance
Coupled plasma mass spectrometry detects manganese content.The size and shape of transmission electron microscope observing MnEMNPs.Electron spin resonanceapparatus detects
The electron spin resonance signal of MnEMNPs.Raman spectrometer detects the raman spectral signal of MnEMNPs.Using the X of scanning electron microscope
Ray Energy Spectrum Analysis analyzes the element composition of MnEMNPs.X-ray photoelectron spectroscopy analyzes the valence of manganese in MnEMNPs
State.The Detection of Stability of Mn in MnEMNPs：By MnEMNPs solution (100 μ g Mn mL-1, triplicate) and it is added in vial,
It is stored at room temperature.In different time points, (17500 revs/min, 15 minutes) are centrifuged, takes 200 μ L supernatants, chloroazotic acid resolution, inductive coupling
Plasma mass spectrometry detects manganese content.
The results show that compared with levodopa and potassium permanganate, the absorption spectrum of reaction solution shows extensive optics
It absorbs, and with the extension in reaction time, absorbance constantly increases, the reason is that caused by levodopa oxidation, polymerization, referring to
Fig. 2.The MnEMNPs aqueous solutions of acquisition can be kept six months or more, macroscopic heavy poly- without occurring, and show it with excellent
Water dispersible.
MnEMNPs is shown as intimate monodispersed spherical structure on transmission electron microscope picture, and size is more uniform, and size is in 80-
100nm or so.Due to the interaction between oligomer subunit, high magnification transmission electron microscope picture shows that MnEMNPs is highdensity
Close stratiform stacking provisions.On electron spin resonance spectroscopy figure, MnEMNPs shows the wide list of the characteristic similar with natural black pigment
Row spectrum.The Raman spectrum of MnEMNPs is in 1387cm-1And 1590cm-1Place shows the characteristic letter similar with natural black pigment
Number.Result above prompts successfully synthesis of melanin sample nano particle jointly.Scanning electron microscope (SEM) photograph and elemental analysis (carbon, oxygen, manganese) knot
Fruit confirms that manganese element is uniformly distributed in MnEMNPs.X-ray photoelectron spectroscopic analysis result is shown in 653.65 and 641.5eV
There is two characteristic signal peaks, respectively Mn in place2p1/2And Mn2p3/2, the prompt of X-ray photoelectron spectroscopic analysis result,
Manganese in MnEMNPs exists in the form of the manganic of most bivalent manganese and fraction.Inductively coupled plasma mass spectrometry is examined
It surveys the results show that manganese charging ratio is up to 10.2%, far above document report before.It is being stored at room temperature after a week, manganese content is basic
Free of losses, the manganese ion for improving chelating have high stability.Result above prompts the high MnEMNPs for carrying manganese amount of successfully synthesis.
Referring to Fig. 3.
The magnetic performance of 3.MnEMNPs characterizes：
Vibrating specimen magnetometer detects the magnetic susceptibility of MnEMNPs at room temperature.Fast Field cycle NMR relaxation instrument exists
At room temperature to the proton 1/T of MnEMNPs1And 1/T2Nuclear magnetic resonance dispersion (1H NMRD) situation characterized, and measurement range is
0.09-1.45T (corresponding 4-62MHz protons Larmor frequency).In order to measure longitudinal relaxation rate (r1) and transverse relaxation rate (r2),
By MnEMNPs solution according to concentration gradient doubling dilution to 0-0.5mM, with Gd-DTPA and Mn2+Standard items are as a contrast.In room
Image Acquisition is carried out to solution using 7.0T toys magnetic resonance imager under the conditions of temperature, parameter is as follows：(1) T1RARE sequences：
Repetition time/echo time：1500/8ms；Echo sounding：8ms；Encourage number：4；Thickness：1mm, the visual field：2.5×2.5cm；
Matrix：256×256；(2) T1-map sequences：Repetition time：447ms-5500ms；Echo sounding：8.5ms；Echo sounding：
8.5ms；Echo：10；Thickness：1mm；The visual field：2.5×2.5cm；Matrix：256×256；(3) Turbo RARE-T2 sequences
Row：Repetition time/echo time：2500/35ms；Echo sounding：11.5ms；Encourage number：4；Thickness：1mm, the visual field：2.5×
2.5cm；Matrix：256×256；(4) T2-map MSME sequences：Repetition time：4500ms；Echo time：9.5-237.5ms；
Echo time：9.5ms；Echo：25；Thickness：1mm；The visual field：2.5×2.5cm；Matrix：256×256.r1And r2Relaxation rate
Respectively by analyzing 1/ relaxation time (s-1) linear relationship between concentration of metal ions (mM) obtains.In addition, being respectively adopted
9.4T toys magnetic resonance device (Bio-Spec, Brooker, Germany), 3.0T clinical magnetic resonances instrument (Magnetom Verio, west gate
Son, Germany), the desk-top relaxation instrument of 1.5T (HT-MICNMR-60, extensive region is red, China) and 0.5T nuclear magnetic resonance spectroscopy imaging systems
MRI relaxation rates under (NMI20, Newman, China) different field strength of detection.
Vibrating specimen magnetometer testing result shows the magnetic susceptibility of MnEMNPs at room temperature as the field strength of applied field increases
And increase, without apparent coercivity and remanent magnetism, prompt MnEMNPs is paramagnetism.The saturated magnetization rate of MnEMNPs is very low
(0.66emu g-1), the T for prompting the local magnetic field generated very faint and minimum2Attenuation effect.A variety of coordination mechanism result in
Magnetic interaction in different coordination environments and different particles so that1The shape of H NMRD is relative complex.The relaxation of MnEMNPs
Henan rate increases, r with the increase of proton Larmor frequency1Relaxation rate and r2Relaxation rate reaches maximum in 42.5 and 55MHz respectively
Value.1The shape and amplitude of H NMRD prompts at least part of metal ion center and more than one hydrone phase interaction
With referring to Fig. 4.
When concentration of metal ions is fixed, MnEMNPs is shown than Gd-DTPA and Mn under identical field strength2+Standard items are more
Good T1Comparison.Under 1.5T field strength, the r of MnEMNPs1Relaxation rate is up to 60.8mM-1s-1, it is 9 times or so of clinical gadolinium agent, is
Mn2+8 times of standard items.The r of MnEMNPs1Melanin of the relaxation rate far above most metal ion mixings of document report
Sample nano particle and MRI nanometer contrast medium based on manganese.Rs of the MnEMNPs under 1.5,3.0 and 7.0T field strength2Relaxation rate is distinguished
It is 52.2,82.1 and 145.4mM-1s-1。
Table 1 is MnEMNPs, Mn under different field strength2+The relaxation rate of standard items and Gd-DTPA are analyzed.When magnetic field intensity from
When 1.5T increases to 9.4T, the r of MnEMNPs2/r1Ratio increases to 10.79 from 0.86.These results prompt MnEMNPs can be used as
High-performance T1-T2Double mode MRI contrast agent, referring to table 1.
Table 1
4. polyethyleneglycol modified and characterization, colloidal stability and blood compatibility detection.
The preparation of polyethyleneglycol modified MnEMNPs (PMnEMNPs)：The polyethylene glycol of MnEMNPs and sulfydryl end are existed
With the mass ratio 1 that feeds intake in alkaline solution (pH=9.8-10.3):5 are mixed, and are stirred overnight under room temperature.Deionized water from
The heart washs three times, to remove extra polyethylene glycol.It is spare that the PMnEMNPs of acquisition is resuspended in deionized water.
The uv-visible absorption spectroscopy of MnEMNPs and PMnEMNPs is detected using all-wave length microplate reader.
Dynamic light scattering detects size of hydration：It takes in 1mL MnEMNPs or PMnEMNPs solution to sample cell, is placed in grain
It spends in analyzer test chamber.Data acquisition conditions are 173 ° of the angle of diffraction, 25 DEG C of test temperature.
Fourier Transform Infrared Spectrometer detects chemical group：Vacuum drying potassium bromide powder is ground with agate mortar
Mill pipettes the sample to be tested after appropriate freeze-drying, by sample and potassium bromide according to about 1：100 mass ratio in mortar again
Mixing, grinding.Lower sheeting sample preparation is irradiated in infrared baking lamp, sample is placed on Fourier infrared spectrograph and is detected.
Colloidal stability detects：By 100 μ g/mL MnEMNPs or PMnEMNPs be dispersed in different physiological solutions (ultra-pure water,
Phosphate buffer, physiological saline, 5% bovine serum albumin(BSA), serum free medium) in, it is stored at room temperature, is incubated 24 hours.It is small
The heart collects 100 μ L supernatant liquids, its uv-visible absorption spectroscopy is detected using all-wave length microplate reader.
Hemolytic experiment：New blood is obtained from healthy Blab/c mouse through eye socket, is placed in anticoagulant tube, is stored at room temperature 30 points
Clock.It 3000 revs/min, centrifuges 10 minutes, it is careful to draw lower layer's red blood cell, it is dense to be diluted to 0.25% volume with phosphate buffer
Degree.The MnEMNPs or PMnEMNPs of dilution and various concentration are incubated 10 hours altogether under the conditions of 37 DEG C.Deionized water or phosphorus
Phthalate buffer is respectively as positive control and negative control.Then, it 15000 revs/min, centrifuges 5 minutes, it is careful to draw 200 μ
L supernatant liquids, using absorbance value at all-wave length microplate reader detection 541nm.
Uv-visible absorption spectroscopy shows that optics of the polyethyleneglycol modified front and back nano particle near infrared region is inhaled
It receives without significant change.Dynamic light scattering result show MnEMNPs and PMnEMNPs average size of hydration be respectively 144nm and
162.5nm.The size of hydration of MnEMNPs is slightly larger than transmission electron microscope size, it may be possible to caused by the swelling effect of polymer.Fu
In leaf transformation the results of FT-IR show, 1082cm-1The characteristic peak at place is derived from the C-O-C keys of polyethylene glycol, prompts MnEMNPs tables
Polyethylene glycol is successfully modified in face.In different physiological solutions, (ultra-pure water, phosphate buffer, physiological saline, 5% ox blood are pure
Albumen, serum free medium) in be incubated 24 hours after, the uv-visible absorption spectroscopy of MnEMNPs shows different degrees of
Decline, and uv-visible absorption spectroscopies of the PMnEMNPs in these solution is consistent substantially, prompts Pegylation can
Improve the colloidal stability of nano particle.Hemolytic test test result shows that 200 μ g/mL MnEMNPs can trigger haemolysis, and phase
PMnEMNPs with concentration has no apparent haemolysis, shows that the blood compatibility of nano particle can be improved in Pegylation.Referring to figure
6。
5. cellular uptake experiment, cytoactive detection and cell MRI
Cellular uptake is tested：By humanized's glioblastoma U87MG cell inoculations in 12 porocyte culture plates, cell is waited for
When being fused to 80% or so, 50 μ g/mL PMnEMNPs are added, are incubated altogether with cell 6 hours.The cell conduct being not handled by
Control.Phosphate buffer wash cell three times, observed, taken pictures by inverted microscope.
Cell transmission electron microscope is tested：By U87MG cell inoculations in 6cm Tissue Culture Dish, wait for cell fusion to 80% or so
When, 50 μ g/mL PMnEMNPs are added, are incubated altogether with cell 6 hours.The cell being not handled by is as a contrast.Digest, centrifuge,
Cell, fixation, sample preparation are collected, transmission electron microscope is observed, taken pictures.
Cellular uptake quantitatively detects：By U87MG cell inoculations in 6cm Tissue Culture Dish, wait for cell fusion to 80% or so
When, the PMnEMNPs of various concentration is added, is incubated altogether with cell 6 hours.The cell being not handled by is as a contrast.Digestion, from
The heart collects cell, and overnight, 0.22 μm of membrane filtration, Inductively coupled plasma mass spectrometry quantitatively divides manganese content for chloroazotic acid digestion
Analysis.
Cytoactive detection：By 5000 U87MG cell inoculations in 96 porocyte culture plates, after cell is adherent, add
The PMnEMNPs for entering various concentration is incubated 24 hours altogether with cell.PMnEMNPs is to cell activity for cell activity experiment detection
It influences.
Cell imaging is tested：U87MG cell inoculations are added in 6cm Tissue Culture Dish when cell fusion is to 80% or so
The PMnEMNPs for entering various concentration is incubated 6 hours altogether with cell.The cell being not handled by is as a contrast.Digestion, is received at centrifugation
Collect cell.1% low melting-point agarose of cell is disperseed, the magnetic resonance T of different grouping cell is respectively compared1、T2Relaxation time.
Referring to Fig. 7, after being incubated 6 hours with 50 μ g/mL PMnEMNPs, optical microscope image shows that U87MG into the cell may be used
See a large amount of dark brown coloured particles, and extracellular background is very clean.Cell transmission electron microscope picture is shown in visible in endosome structure
Nano particle exists, and shows that PMnEMNPs can effectively be absorbed by U87MG cells.After being incubated with various concentration PMnEMNPs, U87MG
Cell activity shows that PMnEMNPs has good biocompatibility without being substantially reduced.Inductively coupled plasma mass spectrometry shows,
U87MG cells can effectively absorb PMnEMNPs, and show concentration dependent.After being incubated with the PMnEMNPs of various concentration,
U87MG cells show good positivity and negativity Contrast enhanced effect simultaneously.The T of cell1Relaxation time and T2Relaxation time is aobvious
Writing reduces, and is in concentration dependent, prompts the PMnEMNPs being ingested that cell visualization degree can be improved.
6. tumor-bearing mice MRI
PMnEMNPs is being injected with 20mg/kg mouse weights dosage after tail vein injects U87MG mice with tumor respectively
Before, injection after different time points to the row magnetic resonance imaging of mouse tumor position.Specific imaging parameters are as described above.Comparison of tumor portion
Position different time points signal and relaxation time situation of change.
Referring to Fig. 8, after tail vein injection PMnEMNPs, U87MG mice with tumor is in T1Weighted image and T2Divide on weighted image
Positivity and negativity Contrast enhanced effect are not shown, reach peak value within 2 hours after injection.In order to eliminate between different mice with tumor
Difference, by the T of tumor locus1Relaxation time and T2Relaxation time carries out quantitative analysis, by the tumour portion before injection PMnEMNPs
The position relaxation time is defined as 100%.The results show that the T of tumor locus1Relaxation time and T2Relaxation time significantly reduces, and is injecting
Reach within 2 hours peak value, the T of the tumor locus of this time point afterwards1Relaxation time and T2Relaxation time be respectively inject PMnEMNPs it
Preceding 70% and 85%.Hereafter, tumor locus signal and relaxation time gradually restore, 24 hours after injection, tumor locus
T1Relaxation time and T2Relaxation time is respectively 86% and 91% before injecting PMnEMNPs.Internal MRI the result shows that
PMnEMNPs can be effectively enriched with by high penetration and high retention effect in tumor locus, and tumor locus contrast is effectively improved.
7. biocompatibility detects
Biochemical index：PMnEMNPs is injected into healthy mice through tail vein with 20mg/kg mouse weights dosage, is not noted
Penetrate PMnEMNPs groups mouse as a contrast.Different time points (3 days, 7 days, 14 days) after injection, using biochemical index instrument
Quantitative detection is carried out to Biochemical Indices In Serum.Testing index includes albumin, glutamic-pyruvic transaminase, glutamic-oxalacetic transaminease, alkaline phosphatase
Enzyme, total protein, serum creatinine, uric acid, urea etc..
Histotomy hematoxylin eosin staining：PMnEMNPs is injected with 20mg/kg mouse weights dosage through tail vein and is good for
Health mouse does not inject PMnEMNPs groups mouse as a contrast.The 15th day after injection, 10% chloral hydrate anesthesia mouse.First use
50mL physiological saline is perfused after heart rinses blood, then with 4% paraformaldehydes of 50mL.Be rapidly separated and obtain the heart, liver, spleen,
The main organs such as lung, kidney are soaked in 10% formalin.Paraffin embedding is carried out after 24 hours.It is cut with 5 microns of thickness
Piece, dewaxing.Histotomy immerses in hematoxylin dye liquor 10 minutes, eosin stains 5 minutes, and inverted microscope observation is taken pictures.
Mouse weight monitors：PMnEMNPs is injected into healthy mice through tail vein with 20mg/kg mouse weights dosage, is not noted
Penetrate PMnEMNPs groups mouse as a contrast.A mouse weight is every other day weighed, is monitored 15 days altogether.
Referring to Fig. 9, each Testing index no significant difference of experimental group and control group prompts PMnEMNPs without apparent liver kidney poison
Property.
Referring to Figure 10, the results show that each cells of organs morphology of experimental group and control group has no notable difference, prompt
PMnEMNPs is without apparent toxicity in vivo.
Referring to Figure 11, the results show that the mouse weight of experimental group and control group has no notable difference, prompt PMnEMNPs without
Apparent toxicity in vivo.
Result above prompts PMnEMNPs to have good biocompatibility.
Conclusion：
The present patent application is by adulterating strategy successfully MnEMNPs of the synthesis with superior water dispersibility in novel polymerization.
Due to have it is high carry manganese amount (>10%) and geometry restriction effect, the MnEMNPs of acquisition have good T1-T2MRI pairs of double mode
Than enhancing ability, under 1.5T field strength, r1Up to 60.8mM-1s-1.After tail vein injects mice with tumor, MnEMNPs shows excellent
Different tumour T1-T2Double mode MRI effects have vast potential for future development in terms of improving tumor imaging contrast.System of the present invention
Standby process is easy, mild, green, and repeatability is high, and manganese ion and melanin are naturally occurring in vivo, can be had by organism
Effect metabolism has greatly clinical conversion value.
Claims (6)
1. a kind of high melanin sample nano material for carrying manganese amount and high relaxation rate, which is characterized in that the material is by manganese source and oxidation
Agent, levodopa are polymerized as precursor molecule, use potassium permanganate as manganese source and oxidant, for water-dispersed, load
Manganese amount>10% and relaxation rate under 1.5 T field strength,R 1 For 60.8 mM-1s-1The melanin sample nano particle of additive Mn
MnEMNPs, the nano material are made using following methods：
By levodopa suspension heating water bath to 50 DEG C, the liquor potassic permanganate of advance ultrasonic disperse is taken, in intense agitation
Under be added rapidly in levodopa suspension, reaction under intense agitation carry out 6 hours, high speed centrifugation be resuspended for several times, will
The MnEMNPs that grain size is 80-100 nm spherical structures is obtained to be scattered in deionized water.
2. height carries the preparation method of the melanin sample nano material of manganese amount and high relaxation rate, which is characterized in that the material is moisture
It is scattered, carry manganese amount>10% and relaxation rate under 1.5 T field strength,R 1 For 60.8 mM-1s-1The melanin sample nano particle of additive Mn
MnEMNPs, preparation method has following steps：
By levodopa suspension heating water bath to 50 DEG C, the liquor potassic permanganate of advance ultrasonic disperse is taken, in intense agitation
Under be added rapidly in levodopa suspension, reaction under intense agitation carry out 6 hours, high speed centrifugation be resuspended for several times, will
The MnEMNPs that grain size is 80-100 nm spherical structures is obtained to be scattered in deionized water.
3. preparation method according to claim 2, it is characterised in that：Levodopa：The molar ratio of potassium permanganate is 1：
0.3。
4. preparation method according to claim 2, it is characterised in that：It is 4-5 times that the high speed centrifugation, which is resuspended, centrifugal speed
It is 17500 revs/min, centrifuges 15 minutes.
5. the high melanin sample nano material for carrying manganese amount and high relaxation rate described in claim 1 is in preparation magnetic resonance imaging contrast
Application in agent.
6. application according to claim 5, it is characterised in that：The imaging contrast isT 1-T 2Double mode MRI contrast agent.
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