CN102397565A - Target-dextran-USPIO (Ultra-small Superparamagnetic Iron Oxide) compound particle and preparation method and application thereof - Google Patents
Target-dextran-USPIO (Ultra-small Superparamagnetic Iron Oxide) compound particle and preparation method and application thereof Download PDFInfo
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- CN102397565A CN102397565A CN2011103413939A CN201110341393A CN102397565A CN 102397565 A CN102397565 A CN 102397565A CN 2011103413939 A CN2011103413939 A CN 2011103413939A CN 201110341393 A CN201110341393 A CN 201110341393A CN 102397565 A CN102397565 A CN 102397565A
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Abstract
The invention discloses a target-dextran-USPIO (Ultra-small Superparamagnetic Iron Oxide) compound particle and a preparation method and application thereof. The target-dextran-USPIO compound particle takes a USPIO as a core, is coated with dextran on the outer layer, and is marked as Dextran-USPIO; and target-network Dextran-USPIO is formed by reacting hydroxyl on monomolecular dextran with epoxy chloropropane, covalently binding the dextran with an epoxy group through an ether link, reacting the epoxy group on the dextran with hydroxyl on another monomer dextran and crosslinking the dextran on the outer layer into a network polymer. A copolymer with a net like structure is formed by dextran on the surface of the USPIO, so that the stability is enhanced, and the surface hydrophily is greatly improved; as proved by experimental research, the CLN (Cross-linked Network)-Dextran-USPIO has higher surface hydrophily, and the stability in a water phase is remarkably improved; in the presence of a network dextran cross-linked dextran modification layer, the compound particle and tumor cells have higher binding capabilities; and as proved by a tumor-bearing nude mouse experiment, the target-dextran-USPIO compound particle has a potential application value on the aspect of MRI (Magnetic Resonance Imaging ) diagnosis of tumors.
Description
Technical field
The present invention relates to a kind of targeted oxidative ferrum nanoparticle, especially a kind of targeting-glucosan iron oxide nano-granule with better water stability, better surface hydrophilicity; The invention still further relates to the manufacturing approach and the application thereof of this type of nanoparticle.
Background technology
NMR-imaging (Magnetic resonance imaging; MRI) be a complete non-invasive inspection; Its safety is good, does not have ionizing radiation, and the high-resolution anatomic image of any position of patient's whole body, any direction (sagittal plane, coronalplane, transverse section), any tomography can be provided apace; Definition considerably beyond computed tomography (Computer AssistedTomography, CT).MRI is applied to human body brain, nervous system, abdominal part and angiocardiography; Detecting of tissue necrosis, ischemia and multiple malignant tumor is effective especially for detecting; Can satisfy the requirement of focus early diagnosis; Be regarded as a kind of diagnostic method that has much potentiality, in the clinical medicine imaging diagnosis, obtain using widely rapidly.
The SPIO contrast agent is superparamagnetic iron oxide particle, and (Superparamagnetic Iron Oxide SPIO), is a kind of nucleocapsid structure, is examined core and encapsulated shell by superparamagnetism to constitute.Encapsulate shell and be made up of hydrophilic materials such as polysaccharide, lipid and surfactants, the nuclear core is γ-Fe
2O
3Or Fe
3O
4Nucleus, the scale size of nucleus have determined the magnetism characteristic and the MRI efficient of SPIO contrast agent.Particle size range is the USPIO of 10-30nm, and the iron oxide core diameter is merely 6～10nm, and major part is a single domain grain oxidation ferrum.USPIO is little because of particle diameter; Reduced reticuloendothelial system engulfing in liver, the spleen to a certain extent to it; Make plasma half-life significantly prolong, guaranteed effective binding time and the absorption time of tumor focus, can be used as angiocardiography agent and blood pond contrast agent USPIO; Again because USPIO is more by Monocytes picked-up in lymph node tissue and the myeloid tissue, therefore can be used for tumor lymphatic knot MET and detect simultaneously.
Outer shell assembling ligands specific molecule at SPIO; Be called targeting SPIO; Part has antibody, polypeptide ligand, micromolecule part etc.; By means of receptor-ligand specificity combination, targeting USPIO (microminiature SPIO) can with the specific surfaces receptors bind of in-vivo tissue or organ, thereby reach the purpose of particular target cell, tissue, organ being carried out the MRI Enhanced Imaging.The AG-USPIO that is equipped with arabinogalactan like outer surface can combine with the asialoglycoprotein receptor of surface of hepatocytes specifically, and does not combine with other histiocyte, reaches the purpose of liver specificity MRI Enhanced Imaging.The anti-VCAM-1-targeting SPIO that adopts the selectively targeted peptide of blood vessel adhesion molecule-1 (VCAM-1) to make up can detect the Atheromatosis speckle in early days.Targeting property USPIO is current and the main research and development focus of SPIO in the future; Because the initiative special target effect that it had; Range of application will be by expansion greatly; Can develop into the targeting MRI contrast agent of specific cells, tissue, organ, as tumor focus detect, the Atheromatosis speckle is detected, grow spike, cell marking etc. in the stem cell transplantation body.
The glucosan finishing SPIO of conventional method preparation, because of glucosan is to rely on the surface physics adsorption to be wrapped on the SPIO core, so the glucosan surface is prone to come off from the surface, causes glucosan SPIO poor stability in physiological solution, is not suitable for long preservation.
Summary of the invention
The present invention is in order to solve the problem that exists in the prior art; A kind of targeting-glucosan-USPIO compound particle is provided; Glucosan has formed on the USPIO surface has cancellated copolymer; Not only stability strengthens, and is more suitable for polypeptide marker, and the present invention also provides the manufacturing approach and the application thereof of the iron oxide nano-granule of this type of cross-linked network glucosan finishing.
Targeting-glucosan of the present invention-USPIO compound particle, said targeting-glucosan-USPIO compound particle is core with USPIO, outer dextran-coated is designated as Dextran-USPIO; Hydroxyl on the unimolecule glucosan and epichlorohydrin reaction; Through ehter bond epoxy radicals on covalent bond on the glucosan; Hydroxyl reaction on epoxy radicals on the said glucosan and another unimolecule glucosan; With said outer field glucosan crosslinked be network polymers, form targeting-netted Dextran-USPIO, be designated as targeting-CLN-Dextran-USPIO.
Preferably: between said USPIO and glucosan, also coat one deck enuatrol.
Preferably: the core particle diameter of said USPIO is 5-10nm, and the particle diameter of said targeting-CLN-Dextran-USPIO is 15-50nm.
Preferably: the core particle diameter of said USPIO is 6-8nm, and the particle diameter of said targeting-CLN-Dextran-USPIO is 20-30nm.
Preferably: the targeting of said compound particle is a kind of among P1:GARYCRGDCFDG, P4:ALNGREESP, P6:ARYCRGDCFDALNGREESP, the P8:GAPPRGALNGREESP.
Adopting epoxychloropropane to impel the surperficial glucosan of Dextran-USPIO crosslinked is network polymers, introduces active epoxy radicals on the Dextran-USPIO surface simultaneously, so that the connection of target polypeptide.Thereby improved Dextran at Fe
3O
4The combination fastness of particle surface, the surface hydrophilicity of enhancing USPIO is strengthened the dispersion stabilization of compound particle in aqueous solution, reduces its non-specific adsorption in vivo.
The response mechanism of hydroxyl is in epoxychloropropane and the glucosan:
(1) under priming reaction (primary response) alkali condition epoxychloropropane with hydroxyl reaction on the glucosan, through ehter bond epoxy radicals on covalent bond on the glucosan.The epoxy radicals of this free end can be used as the active reactive group that polypeptide connects, thereby introduces target polypeptide on the CLN-Dextran-USPIO surface.
(2) under cross-linking reaction (side reaction) alkali condition; Epoxy radicals on the glucosan can be with the hydroxyl reaction of another monomolecular glucosan; Thereby with glucosan crosslinked be network polymers, through cross-linking reaction, can make the glucosan cross linked polymer stably be coated on the USPIO surface.
(3) other side reaction-hydrolysis: the long-time down progressively hydrolysis generation glycerol that contacts with alkali liquor of epoxychloropropane high temperature.
Control the occurrence degree of primary response and side reaction through controlling reaction time and pH value; Reduce the generation of side reaction (3); Thereby both guaranteed that USPIO surface glucosan took place crosslinked; Generation has the CLN-Dextran-USPIO of netted glucosan finishing, has kept part active epoxy base again simultaneously, is used for follow-up polypeptide ligand and connects.
Adopt the density of sodium thiosulfate titration epoxy radicals, in the time of 25 ℃, with the priming reaction time be controlled at 4-8 hour proper, the density of epoxy radicals is higher like this, helps the carrying out of next step coupling reaction.
A kind of method of making above-mentioned targeting-glucosan-USPIO compound particle may further comprise the steps:
(1) the USPIO suspension is joined in 15% the glucosan water, under 80 ℃ condition, stirred 60 minutes, the ratio of ferroso-ferric oxide is 1 in glucose and the solution: 1-9: 1, prepare glucosan-USPIO compound particle;
(2) be under the alkali condition of 11-13 at pH value; Glucosan behind the purification-USPIO compound particle and epoxychloropropane are mixed; Wherein, epoxychloropropane: water-based magnetic fluid (v/v) scope is 1: 20-1: 4, and reaction is 2-8 hour under 20-42 ℃ condition; Washing, resuspended obtains the water-based magnetic fluid of the glucosan-USPIO compound particle of epoxy-activated;
(3) buffer and target polypeptide are joined in the water-based magnetic fluid of glucosan-USPIO compound particle of epoxy-activated room temperature concussion reaction 5 hours; Gained is targeting-CLN-glucosan-USPIO compound particle.
Preferably: the ratio of ferroso-ferric oxide is 7: 1 in middle glucose of said step (1) and the solution.
Preferably: reaction temperature is 30 ℃ in the said step (2), and the response time is 3 hours.
Preferably: also can add glycerol after said step (3) reaction finishes, concussion reaction 8-16 hour.
The invention also discloses a kind of tumour-specific MRI image-forming contrast medium, comprise above-mentioned targeting-glucosan-USPIO compound particle.
Investigate the nuclear magnetic resonance effect of targeting CLN-Dextran-USPIO, on the animal level, investigate its plasma half-life, study its bio distribution and metabolism situation of each internal organs in animal body tumor bearing nude mice.The result shows: the plasma half-life of Dextran-USPIO is about 2.8hrs, and the plasma half-life of CLN-Dextran-USPIO then is about 6.2hrs, and the stability of this explanation CLN-Dextran-USPIO obviously improves; Simultaneously, because of the change of its surface hydrophilic performance, can escape engulfing of reticuloendothelial system better, be better tumour-specific image-forming contrast medium.Tumor bearing nude mice nuclear magnetic resonance experiment shows that the MR signal between tumor/non-tumor tissue is 1.83 times, can have bigger using value in the tumor that demonstrates better on the MRI below the 5mm.The tissues of experimental animals of Target-CLN-Dextran-USPIO is distributed and drug metabolism study shows: Target-CLN-Dextran-USPIO can specificity is dense in tumor gathers, and mainly passes through liver, and spleen and kidney carry out metabolism.
The invention has the beneficial effects as follows: the present invention has adopted the epoxychloropropane method to prepare a kind of USPIO (Cross-linked Network-USPIO with the finishing of cross-linked network glucosan; CLN-Dextran-USPIO) compound particle; Glucosan has formed on the USPIO surface has cancellated copolymer; Not only stability strengthens, and is more convenient for and target polypeptide combines.Solved cross-linked network, these two problems of polypeptide marker simultaneously, preferably the later stage has added glycerol again, and the same glycerine reaction of residual epoxy base is combined on the nanoparticle glycerol molecule, thereby further improves the nanoparticle surface hydrophilicity.Show that through experimentation CLN-Dextran-USPIO has better surface hydrophilicity, its stability at aqueous phase is also obviously improved.Adopt blue staining in Prussia and ferrum standard curve determination method to measure the external combination of compound particle of the present invention with tumor cell; Show that Target-CLN-Dextran-USPIO has better binding ability, the tumor bearing nude mice experiment shows that also this kind Target-CLN-Dextran-USPIO has potential using value aspect the tumor MRI image-forming diagnose.
Description of drawings
Fig. 1 is enuatrol-nanometer Fe among the present invention
3O
4Tg-DSC figure.
Fig. 2 is enuatrol-nanometer Fe among the present invention
3O
4Transmission electron microscope photo.
Fig. 3 is enuatrol-nanometer Fe among the present invention
3O
4XRD figure.
Fig. 4 is enuatrol-nanometer Fe among the present invention
3O
4Infrared spectrum.
Fig. 5 is the infared spectrum of glucosan-USPIO among the present invention.
Fig. 6 is the infrared spectrogram of the epoxidation cross-linking reaction of targeting-CLN-Dextran-USPIO among the present invention.
Fig. 7 handles the infrared spectrogram that connects polypeptide for the epoxychloropropane of targeting-CLN-Dextran-USPIO among the present invention.
Fig. 8 is the SEM projection electromicroscopic photograph of targeting-CLN-Dextran-USPIO of the present invention.
Fig. 9 is that the external HUVEC cell of targeting-CLN-Dextran-USPIO of the present invention combines the experiment comparison diagram.
Figure 10 is the iron content comparison diagram after targeting of the present invention-CLN-Dextran-USPIO combines cell.
Figure 11 is the mensuration figure of the plasma half-life of targeting-CLN-Dextran-USPIO of the present invention.
Figure 12-13 is nuclear magnetic resonance and the tissue distribution figure thereof of targeting-CLN-Dextran-USPIO of the present invention in the tumor bearing nude mice body.
The specific embodiment
Below in conjunction with specific embodiment technical scheme of the present invention is further described, but protection scope of the present invention is not limited thereto.
Hereinafter, superminiature SPIO nanoparticle is USPIO (Ultrasmall Superparamagnetic IronOxide); Glucosan-USPIO is Dextran-USPIO; Netted glucosan-USPIO is CLN-Dextran-USPIO (Cross-linked Network-Dextran-USPIO); CLN-Dextran-USPIO after combining with part is targeting-CLN-Dextran-USPIO, also can be designated as Target-CLN-Dextran-USPIO.
Synthesizing of targeted hybrid polypeptide:
Adopt the synthetic polypeptide of solid-phase polypeptide synthetic method, the aminoacid sequence of synthetic 4 target polypeptides is respectively P1:GARYCRGDCFDG; P4:ALNGREESP; P6:ARYCRGDCFDALNGREESP; P8:GAPPRGALNGREESP.Synthesize on the FMOC-Tyr-Wang-Resin resin and accomplish, behind resin swelling and the deprotection, adopt Fmoc protection aminoacid, the HBTU+NMM of secundum legem or DIC+HOBT condensation method are carried out the aminoacid coupling successively and are joined.Cutting washing after condensation is accomplished.Carry out oxidative cyclization after synthetic.The SHIMADZU high performance liquid chromatograph carries out purification.Identify and adopt mass spectrograph.Sample sealing-20 ℃ of preservations in back.The polypeptide P1 that the present invention adopts, P4, P6, P8 buy from Shanghai Pfizer's bio tech ltd.
Instance 2
The preparation of enuatrol-Dextran-USPIO:
At first prepare FeCl
2/ FeCl
3Mixed liquor and NaOH solution, shown in each material consumption following table:
(1) nano ferriferrous oxide of preparation enuatrol monolayer coating:
Adopt high-speed shearing machine to stir, in the glass jacket still of 1L, react, and connect circulating water chennel.Circulating water temperature is adjusted in 70 ℃, connects the import and export of chuck, ON cycle water.With between chuck mouth and the high-speed shearing machine drill bit with preservative film sealing, and feed nitrogen.Rapidly enuatrol, ferrum liquid and alkali liquor are poured in the chuck, opened and stir, good preservative film with rubber belt sealing, keep feeding nitrogen, the air that prevents under high speed shear, to bring into is with the product oxidation.Response time is about 40min, stops to stir and recirculated water, with reaction solution cooling back sucking filtration, is dissolved in the 300ml deionized water after washing 2-3 time.The material that obtains is exactly the nano ferriferrous oxide that the enuatrol monolayer coats, i.e. enuatrol-USPIO.
It characterizes like Fig. 1-shown in Figure 4.Fig. 1 shows Fe for the Tg-DSC figure that thermal analyzer generates
3O
4Surperficial and oily sodium has chemical bond to combine.Second endothermic peak about 650 ℃ is and Fe
3O
4There is the catabolic process of the enuatrol of chemical bond combination on the surface, because the combination of chemical bond is arranged, makes catabolic process postpone.Fig. 2 is JEM-1200EX II type transmission electron microscope TEM, and the nuclear core particle diameter of enuatrol-USPIO is 5-10nm.Fig. 3 is the result of x-ray diffractometer (model is D/MAX 2550VB/PC) test, therefrom can learn Fe in the compound particle
3O
4Consistent with the characteristic peak of its standard card, be cubic crystal structure, through the Fe that coats
3O
4The not change of structure.According to the Scherrer formula: the crystallite dimension that D=κ λ/β cos θ calculates in vertical crystal plane (311) direction is 6.29nm.XRD calculates Fe
3O
4The average-size of crystal grain and tem observation basically identical, this has proved the Fe of preparation
3O
4Particle is a monocrystal.Fig. 4 is the infrared spectrum of oleic acid-USPIO, adopts Nicolet 380 type infrared spectrometers, can find out 570cm among the figure
-1About that occur is Fe
3O
4Characteristic absorption peak, 2920cm
-1And 2850cm
-1Be the characteristic absorption peak of methylene and methyl, 1500cm
-1Near be the characteristic absorption peak of carboxylate.Do not occur in the spectral line enuatrol-C=O (1710cm
-1) absworption peak of structure, in the interpret sample through there not being the enuatrol of residual free state after the washing repeatedly.Carbonyl-C=O in the enuatrol structure all is cleaved into two equal-C-O-singly-bounds, i.e. COO-antisymmetric vibration absworption peak (1600cm
-1) and symmetric vibration absworption peak (1400cm
-1).And drift has taken place in these two absworption peaks in the drawings, corresponding 1545cm
-1And 1405cm
-1Two absworption peaks, this has explained enuatrol and Fe
3O
4The combination of chemical bond is arranged between the particle, formed the structure of the similar carboxylate of triangle.
(2) preparation glucosan enuatrol double-coated nano ferriferrous oxide:
Prepare 15% glucan aqueous solution; Put into and carry out stirring at low speed in the light water bath; Its ferroso-ferric oxide suspension with the above-mentioned enuatrol monolayer coating that obtains is mixed; Adopt common stir about 60min down at 80 ℃, obtain glucosan oleic acid double-coated nano ferriferrous oxide, be designated as: Dextran-USPIO.
According to ferroso-ferric oxide theoretical content ratio in glucosan consumption and the solution, select glucosan: Fe
3O
4Ratio be that 1: 1,3: 1,5: 1,7: 1,9: 1 five different ratios are tested respectively.In the solution of 100ml, make the magnetic solution of five kinds of different proportions, the preparation after with the ultrafiltration pipe under the 2000r/min with distilled water centrifuge washing three times after.The laser light scattering instrument (model is ALV/CGS-5022F) that utilizes German ALV company to produce is measured the size of its hydrodynamics particle diameter; The nano particle size appearance that utilizes Britain MIL company to produce is measured (model is Zetasizer:3000HS) and is tested its Zeta potential, and the result is following:
Can find out that from above result between 5: 1 to 9: 1 ratios, the hydrodynamics particle diameter is controlled at below the 80nm, the Zeta potential absolute value is bigger, belongs to stabilizing solution.Work as glucosan: Fe
3O
4Less than 5: 1 o'clock, the hydrodynamics particle diameter was bigger, and this is because the glucosan good water solubility, and a part is dissolved in the water and is not coated on nanometer Fe
3O
4The surface of particle makes nanometer Fe
3O
4Soft-agglomerated phenomenon is arranged between the particle, make the hydrodynamics particle diameter increase.Simultaneously, the soft-agglomerated Zeta potential absolute value that also can make reduces, and stability of solution descends.And glucosan is crossed and can be made nanometer Fe at most
3O
4Particle surface coats blocked up, and particle diameter increases, and does not utilize the later stage to use.Ratio is that the solution that obtains at 7: 1 is highly stable, and does not have aggregation phenomenon between the particle basically.The infared spectrum of resulting glucosan oleic acid double-coated nano ferriferrous oxide is as shown in Figure 5.
Instance 3
The preparation of targeting-CLN-Dextran-USPIO:
At first prepare CLN-Dextran-USPIO, its step is following:
The H that the water-based magnetic fluid of the Dextran-USPIO compound particle of the 4ml that has prepared is added 4ml
2O dilutes, and is with 10KD ultrafiltration pipe (Mi Libo production) centrifugal ultrafiltration 10min under the 2000rpm rotating speed, with 4mL distilled water ultrafiltration washed twice, resuspended with the 4mL distilled water.
(1) above-mentioned purification Dextran-USPIO water-based magnetic fluid is later added the NaOH that 4ml concentration is 2.5mol/L, mixing, measuring system pH is 13;
(2) add the 0.8ml epoxychloropropane, 30 ℃ of reactions of room temperature 3 hours;
(3) with the ultrafiltration of 10KD ultrafiltration pipe, 5mL distilled water ultrafiltration washing three times, resuspended with the 4ml distilled water, gained is the water-based magnetic fluid of the Dextran-USPIO compound particle of epoxy-activated, is CLN-Dextran-USPIO.
Secondly, preparation targeting-CLN-Dextran-USPIO, its step is following:
(1) get the water-based magnetic fluid of 0.5ml CLN-Dextran-USPIO compound particle, adding 50uL concentration is the polypeptide P6 of 25mmol/L, adds the carbonate buffer solution (pH10) with the isopyknic 0.2mol/L of compound particle, room temperature concussion reaction 5h.
(2) 50% of adding 200ul glycerol, concussion reaction room temperature 8-16 hour.
(3) 10KD ultrafiltration pipe centrifugal ultrafiltration washing, 5mL normal saline washed twice is dissolved in the 0.5ml normal saline at last.Gained is: P6-CLN-Dextran-USPIO.Make P1-CLN-Dextran-USPIO, P4-CLN-Dextran-USPIO, P6-CLN-Dextran-USPIO, P8-CLN-Dextran-USPIO according to above-mentioned described step.
Its infrared spectrum characterization such as Fig. 6-shown in Figure 8:
1010cm among Fig. 6
-1About occur be-characteristic absorption peak of C-O-C-3433.95cm
-1Be the hydroxyl characteristic absorption peak, the characteristic absorption peak of epoxy radicals is at 910cm
-1About, be more weak absworption peak.
1010cm among Fig. 7
-1About occur be-characteristic absorption peak of C-O-C-3413cm
-1The place be hydroxyl perhaps-N-H-key characteristic absorption peak, 1344cm
-1About be the vibration absorption peak of carbon nitrification key.This shows have polypeptide to be connected on the surface of compound particle.
Fig. 8 is a transmission electron microscope photo, and the mean diameter that shows targeting-CLN-Dextran-USPIO is 15-50nm.
Instance 4
The cell in vitro of targeting-CLN-Dextran-USPIO combines experiment:
1. cell culture:
Pulmonary carcinoma A549 (derives from ATCC; NO.CCL-185) and Human umbilical vein endothelial cells HUVEC (derive from ATCC; NO.CRL-1730) cell strain is incubated at DMEM (Dulbecco ' s Modified Eagle Media) culture fluid (containing 10% NBCS, 100U/L penicillin, 100U/L streptomycin), puts 37 ℃, 5% CO
2Constant incubator in.The trophophase cell of taking the logarithm experimentizes, and cell inoculation is in 24 orifice plates.
2. prussian blue staining method characterizes targeting-CLN-Dextran-USPIO and cells in vitro combines
Get CLN-Dextran-USPIO, P1-CLN-Dextran-USPIO, P4-CLN-Dextran-USPIO, P6-Dextran-USPIO, each 50ul of P8-Dextran-USPIO respectively with the A549 cell at 37 ℃, 5% CO
2Constant incubator in hatch 16h.
A. inhale and go culture medium,, decide 30min with 1ml 4% formaldehyde fixed is liquid-solid with PBS buffer washed twice.
B.PBS buffer washed twice adds the Prussian blue dye liquor of 1ml (2% ferrocyanide aqueous solutions of potassium and 2%HCL aqueous solution one to mix) dyeing 10min.
Add eosin stain (0.5% Yihong, 75% ethanol) dyeing 5min after the C.PBS buffer washed twice
Observation by light microscope experimental result after the D.PBS buffer washed twice.
Experimental result is as shown in Figure 9; (Human umbilical vein endothelial cells derives from ATCC to the HUVEC of contrast CLN-Dextran-USPIO group, NO.CRL-1730) invisible basically blue ferrum particulate manifesting in the Cytoplasm; And P1-CLN-Dextran-USPIO; P4-CLN-Dextran-USPIO, P6-CLN-Dextran-USPIO, the HUVECs of P8-CLN-Dextran-USPIO can see has tangible blue ferrum granule.
3. detect Dextran-USPIO, Target-Dextran-USPIO and cell binding ability
At first titer preparation:
With 70.2mg (NH4)
2Fe (SO4)
2.6H
2O adds 3.5ml 10mol/L concentrated hydrochloric acid, adds water and is settled to 100ml, and this moment, the concentration of Fe2+ was 1.791mmol/L.Get 1ml, add water and be settled to 80ml, obtain the Fe titer, the concentration of Fe2+ is 22.387 μ mol/L.
Get ferrum titer 0,0.8,1.6,2.4,3.2,4ml respectively in the scale test tube, label is that to add the entry standardize solution in 1,2,3,4,5,6 six test tube respectively be 4ml, and adds boiling water bath 5min behind 0.2ml 10% oxammonium hydrochloride..In each test tube, add 0.1mol/L acetic acid-sodium acetate buffer 0.5ml again, 0.5% adjacent luxuriant and rich with fragrance sound of vomiting quinoline 0.2ml, shake up static, treat that it fully develops the color after, being 0 to measure in each test tube liquid in No. 1 test tube at the absorbance at 510nm place.Draw concentration-absorbance standard curve, as shown in the table:
| Concentration (μ mol) | 0 | 4.477 | 8.955 | 13.433 | 17.910 | 22.387 |
| |
0 | 0.043 | 0.093 | 0.143 | 0.191 | 0.238 |
Cell combines the measuring iron content:
The A549 cell inoculation is in 6cm culture dish (2 * 10
5/ hole); 37 ℃, 5%CO2 incubator were cultivated 24 hours, removed culture fluid, added the DMEM culture fluid 2ml that does not contain serum in each hole; Add non-target-Dextran respectively; P1-Dextran-USPIO, P4-Dextran-USPIO, incubated overnight in P6-Dextran-USPIO, the back 37 ℃ of incubators of each 50 μ l of P8-Dextran-USPIO (5 in multiple hole).Culture fluid is removed in suction, normal saline washing 2 times, and every hole adds 200 μ l6mol/L HCl and dissolves iron ion; Add boiling water bath 5min behind 0.1ml 10% oxammonium hydrochloride. respectively, all Fe3+ are reduced to Fe2+, add 0.1ml acetic acid-sodium acetate buffer; Make the pH of system be about 9, add the adjacent luxuriant and rich with fragrance sound of vomiting quinoline of 0.1ml0.5%, shake up and leave standstill 10min; After treating fully colour developing, take the aqueous solution as the absorbance at blank determination 510nm place.
Experimental result is as shown in Figure 9: compare with non-Target-CLN-Dextran; P1-CLN-Dextran-USPIO; P4-CLN-Dextran-USPIO; P6-CLN-Dextran-USPIO, P8-CLN-Dextran-USPIO have the specificity combination with the A549 lung adenocarcinoma cell line, and be the strongest with the P6-CLN-Dextran-USPIO binding ability especially.Explain that target polypeptide successfully is connected in the CLN-Dextran-USPIO surface, and have cell combination activity.
Embodiment 6
Targeting-CLN-Dextran-USPIO is to the nuclear magnetic resonance and the tissue distribution research thereof of tumor bearing nude mice: investigate the nuclear magnetic resonance effect of targeting CLN-Dextran-USPIO to tumor bearing nude mice; On the animal level, investigate its plasma half-life, study its bio distribution and metabolism situation of each internal organs in animal body.
Key step and result thereof:
One. set up the A549 model of nude mice bearing tumor.
4 the week age Balb/c strain nude mice, according to 10
7Cell/ml tucks in subcutaneous vaccination human lung adenocarcinoma A549 cell strain 0.1ml in the right side, aseptic condition was raised down after 25-35 days, and tumor growth is to the 2-3mm size.
Two. the targeting CLN-Dextran-USPIO radiography medicine of test
Comprise: (1) Non-Target-CLN-Dextran-USPIO; (2) P1-CLN-Dextran-USPIO; (3) P4-CLN-Dextran-USPIO; (4) P6-CLN-Dextran-USPIO; (5) P8-CLN-Dextran-USPIO.
The plasma half-life of three .Dextran-USPIO and CLN-Dextran-USPIO is measured
Get 6 ages in week 9 of normal female Kunming mouses; Be divided into three groups at random; Every group 3: normal saline matched group, Dextran-USPIO group and CLN-Dextran-USPIO group; Freely drink water and fasting 12h after, each group is tail vein injection 100uL normal saline and 0.5mg/100uL Dextran-USPIO and 0.5mg/100uL CLN-Dextran-USPIO respectively.Respectively at injection back 15min, 1hr, 3hrs, 6hrs, 9hrs, 12hrs, 15hrs, 24hrs cut the about 100uL of tail blood sampling in the EP pipe, and 5000rpm*10min is centrifugal, gets 50uL serum, measures serum levels of iron content.
The result is shown in figure 13: the blood plasma concentration of iron of the control group mice of injecting normal saline is in the 19.5-28.5umol/g scope; In the Dextran-USPIO injection back 15min to 3hrs, the blood plasma concentration of iron descends rapidly, and 3 hours blood plasma concentration of iron are 37.8 ± 4.1umol/g; In 6-15 hour; The blood plasma concentration of iron is progressively stable to descend, and drops to 15.1umol/g after 24 hours, is about 2.8hrs through the plasma half-life that calculates Dextran-USPIO.And after the CLN-Dextran-USPIO injection; PC descends and is considerably slower than Dextran-USPIO; Only dropped to 51.2 ± 5.9umol/g in 3 hours, in 6-15 hour, the blood plasma concentration of iron slowly descends; Drop to 13.7 ± 1.9umol/g after 24 hours, be about 6.2hrs through the plasma half-life that calculates CLN-Dextran-USPIO.The raising of CLN-Dextran-USPIO owing to stability and surface hydrophilicity is described, further reduced the phagocytosis of endothelium reticular system, prolonged plasma half-life, for the raising of tumor imaging effect is laid a good foundation.
Four. targeting-CLN-Dextran-USPIO is to the nuclear magnetic resonance of tumor bearing nude mice
Tumor bearing nude mice is respectively through the contrast of tail vein injection 100uL normal saline; 0.5mg/100uL CLN-Dextran-USPIO and 0.5mg/100uL P1-CLN-Dextran-USPIO; P2-CLN-Dextran-USPIO; P6-CLN-Dextran-USPIO, 24hrs carries out the T2WI nuclear magnetic resonance after the administration.
Nuclear magnetic resonance: tumor-bearing mice lumbar injection 1% pentobarbital sodium 0.15ml anesthesia, form images in the enterprising line scanning of PHILIP 1.5T NMR behind the fixation postures.Adopt sweep parameter: SE-T2WI, TR4000ms, TE105ms, FOV12mm, slicethickness, 0.8mm, gap, 0.2mm.Observe distribution, the metabolism situation of various targeting CLN-Dextran-USPIO in tumor and major organs.Adopt ANOVA analysis that the T1 signal strength analysis is carried out in visual ROI zone, research CLN-Dextran-USPIO congregational rate and metabolism situation in tumor.
System thinking above-mentioned various Target-CLN-Dextran-USPIO radiography medicines in intravital distribution of mice and metabolism situation.Estimated the diagnostic value of above-mentioned Target-CLN-Dextran-USPIO radiography medicine, confirmed the using value of radiography medicine MRI image-forming diagnose tumor minimal disease to the MRI imaging of tumor.
Imaging results such as Figure 11, shown in Figure 12: the AO of injecting the tumor of physiological control mice is 0.130 ± 0.016, and the AO of injecting no target CLN-Dextran-USPIO is 0.1660.021, and P1; P2, P6, the AO of P7 targeting CLN-Dextran-USPIO is respectively: 0.225 ± 0.017; 0.179 ± 0.0177; 0.4460.041,0.277 ± 0.037, all be significantly higher than normal saline matched group and no target group.Be illustrated in the tumor bearing nude mice body, the CLN-Dextran-USPIO of targeting all can specificity is dense in tumor gathers.Wherein P6-CLN-Dextran-USPIO has the best dense effect of gathering, and improves 3.43 times than normal saline group signal intensity, improves 2.68 times than no target nanoparticle.The result shows that targeting CLN-Dextran-USPIO can be gathered in tumor locus by specificity, is a kind of tumour-specific MRI contrast agent of function admirable.
Claims (10)
1. targeting-glucosan-USPIO compound particle, it is characterized in that: said targeting-glucosan-USPIO compound particle is core with USPIO, and outer dextran-coated is designated as Dextran-USPIO; Hydroxyl on the unimolecule glucosan and epichlorohydrin reaction; Through ehter bond epoxy radicals on covalent bond on the glucosan, the hydroxyl reaction on the epoxy radicals on the said glucosan and another unimolecule glucosan, with said outer field glucosan crosslinked be network polymers; Again through the surperficial epoxy radicals of cross-linked network glucosan; Target polypeptide in the connection forms targeting-netted Dextran-USPIO, is designated as targeting-CLN-Dextran-USPIO.
2. compound particle according to claim 1 is characterized in that: between said USPIO and glucosan, also coat one deck enuatrol.
3. compound particle according to claim 1 is characterized in that: the core particle diameter of said USPIO is 5-10nm, and the particle diameter of said targeting-CLN-Dextran-USPIO is 15-50nm.
4. compound particle according to claim 3 is characterized in that: the core particle diameter of said USPIO is 6-8nm, and the particle diameter of said targeting-CLN-Dextran-USPIO is 20-30nm.
5. compound particle according to claim 1 is characterized in that: the targeting of said compound particle is a kind of among P1:GARYCRGDCFDG, P4:ALNGREESP, P6:ARYCRGDCFDALNGREESP, the P8:GAPPRGALNGREESP.
6. method of making targeting-glucosan as claimed in claim 1-USPIO compound particle is characterized in that: may further comprise the steps:
(1) the USPIO suspension is joined in 15% the glucan aqueous solution, under 80 ℃ condition, stirred 60 minutes, the ratio of ferroso-ferric oxide is 1 in glucose and the solution: 1-9: 1, prepare glucosan-USPIO compound particle;
(2) be under the alkali condition of 11-13 at pH value; Glucosan behind the purification-USPIO compound particle and epoxychloropropane are mixed; Wherein, epoxychloropropane: water-based magnetic fluid (v/v) scope is 1: 20-1: 4, and reaction is 2-8 hour under 20-42 ℃ condition; Washing, resuspended obtains the water-based magnetic fluid of the glucosan-USPIO compound particle of epoxy-activated;
(3) buffer and target polypeptide are joined in the water-based magnetic fluid of glucosan-USPIO compound particle of epoxy-activated room temperature concussion reaction 5 hours; Gained is targeting-CLN-glucosan-USPIO compound particle.
7. method according to claim 5 is characterized in that: the ratio of ferroso-ferric oxide is 7: 1 in middle glucose of said step (1) and the solution.
8. method according to claim 5 is characterized in that: reaction temperature is 30 ℃ in the said step (2), and the response time is 3 hours.
9. method according to claim 5 is characterized in that: also can add glycerol after said step (3) reaction finishes, concussion reaction 8-16 hour.
10. a tumour-specific MRI image-forming contrast medium comprises like each described targeting-glucosan of claim 1-5-USPIO compound particle.
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