CN104645358A - Pancreatic cancer-targeted nanoscale ultrasonic contrast agent and preparation method and application thereof - Google Patents
Pancreatic cancer-targeted nanoscale ultrasonic contrast agent and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of medicines, in particular to a pancreatic cancer-targeted nanoscale ultrasonic contrast agent and a preparation method and application thereof, and provides a pancreatic-cancer-targeted nanoscale ultrasonic contrast agent. The pancreatic cancer-targeted nanoscale ultrasonic contrast agent is prepared by coating perfluorocty bromide with a polyethylene glycol modified polylactic acid-glycolic acid copolymer and connecting with a pancreatic cancer targeted Hedgeho monoclonal antibody. The invention further provides a preparation method of the nanoscale ultrasonic contrast agent, and an application of the nanoscale ultrasonic contrast agent in preparation of a pancreatic carcinoma diagnosis reagent. The nanoscale ultrasonic contrast agent provided by the invention is designed to specifically target the pancreatic cancer cells for high expression of Hedgeho in early diagnosis of the pancreatic cancer, so as to become an ultrasonic contrast agent with enhanced ultrasound contrast and high specific imaging capability for early diagnosis of the pancreatic cancer.
Description
Technical field:
The present invention relates to medical art, be specifically related to nanoscale ultrasound contrast agents of a kind of cancer of pancreas targeting and its preparation method and application.
Background technology:
Cancer of pancreas onset is hidden, progress fast, treatment difficulty and mortality rate all very high, be classified as " the obstinate fort of 21 century " by medical circle, the sickness rate of recent domestic cancer of pancreas obviously rises.The most early Radical surgery excision in treatment of pancreatic cancer means, when cancer of pancreas major diameter is less than lcm, pathological changes is limited to pancreatic duct endothelium, and root value criterion 5 years survival rates are close to 100%; And major diameter more than lcm time, how with the infringement of the surrounding tissues such as lymph node, blood vessel and pancreas peplos, within postoperative 5 years, survival rate is about 17%, and has the patient of 85% to there will be recurrence and hepatic metastases, mean survival time (MST) 15-19 month.Limitting by conditions for diagnostics, the Resection Rate of current cancer of pancreas is only 10%-20%, and within 5 years, survival rate is only 0.4%-3.4%.Therefore, early diagnosis and early treatment are the keys improving cancer of pancreas prognosis.
Ultrasonic contrast strengthens (contrast-enhanced endoscopic ultrasound, CE-EUS) technology is one that development in recent years is got up can carry out " imaging of blood pond " new technique to focus, to compare other Imaging Methods (as CT, MRI etc.), have cheap, safe, easy to operate, observe the advantages such as real-time, radiationless.CE-EUS is mainly through intravenous injection microvesicle state contrast agent, scanning object interface echo acoustic impedance difference, by tumor focus from surrounding tissue saliency out, in diagnosis of pancreatic cancer, as the conventional means of cancer of pancreas examination, can become one of focus of pancreatic diseases diagnosis research.Because common contrast agent concentration entirety in pancreatic tissue is on the low side, the half-life is short, and less focus there will be fails to pinpoint a disease in diagnosis, and makes this technology have certain limitation.
In recent years, molecular imaging high development, targeted molecular technology is more and more subject to the attention of researcher.Hedgehog albumen is almost expressed at all cancer of pancreas camber, and height expression status occurs in early days at neoplastic lesion, and Hedgehog albumen distributes limited in the normal tissue, there is no the acoustic contrast agent of targeting Hedgehog albumen at present, therefore prepare a kind of can the acoustic contrast agent of targeting Hedgehog albumen cancer of pancreas early diagnosis being had very important significance.
PLGA (polylactic-co-glycolic acid block copolymer) is through the Biodegradable material that U.S. FDA certification can be used for human body, has good hydrophilic and biocompatibility, can be prepared into the nano_scale particle carrier of multi-medicament.Wherein, the technology of PEG (Polyethylene Glycol) is used to modify PLGA nano-carrier comparatively ripe and stable.Research confirms, utilizes PLGA and NH
2after-PEG-COOH prepares new PLGA derivant, target antibody can be anchored in nanoparticle with the-PEG-COOH end reaction of nano-particle surface, forms the nanoparticle with targeting.In addition, nano-particle surface can reduce non-specific the engulfing that nanoparticle is subject to RES (reticuloendothelial system) further after PEG modifies, thus reaches the effect of " stealth ", can reach the longer half-life in body circulation.
The contrast agent being mainly used in ultrasonic contrast enhancing (CE-EUS) is at present mainly microbubble contrast agent.Microbubble contrast agent can the clear display Abnormal Perfusion region of causing due to vascular problem or lesion tissue, thus provide accurate, dynamic perfused tissue information for clinical, but, still there is a lot of problem in this microbubble contrast agent, as: 1. affinity be there is no to pathological tissues, non-specific development can only be carried out to pathological tissues, cannot lesion nature be judged; 2. microbubble diameter relatively large (mostly being 2-6 μm of level), although freely can pass through pulmonary circulation, cannot arrive target cell by penetration rate of blood tube wall; 3. the gas comprised in microvesicle itself has the feature of inborn reflex and backscattering, usually makes the contrast of observed object and background not strong; 4. the half-life is short, and effective Enhanced time is tens of second or several minutes only, the pathological changes checked needing the long period, can only pass through repeatedly bolus administration of contrast agent and just can meet the demands.Liquid fluorocarbon is different from the ultrasound microbubble contrast agent of inborn reflex and backscattering feature, its performance producing ultrasonic enhancing development is based on targeting is assembled: when liquid fluorocarbon contrast agent microsphere is in dispersity, very weak to ultrasonic sensitive, only have when particles agglomerate is to target tissue or cell membrane, just in ultrasonic lower development.This gathering development does not have sound shadow phenomenon and the background noise of microcapsular ultrasound contrast agent, thus can greatly improve resolution accuracy, and perfluoro bromide octane (PFOB) is the representative compound in liquid fluorocarbon contrast agent.
There is no the pertinent literature report of the nanoscale ultrasound contrast agents of targeting Hedgehog albumen at present.
Summary of the invention:
The object of the invention is to solve current cancer of pancreas ultrasonic contrast and to strengthen in (CE-EUS) technology the limitation such as specificity visualization capabilities difference, a kind of nanoscale ultrasound contrast agents of cancer of pancreas targeting is provided.
Another object of the present invention is to provide the preparation method of the nanoscale ultrasound contrast agents of this cancer of pancreas targeting.
The third object of the present invention, the nanoscale ultrasound contrast agents being to provide this cancer of pancreas targeting is preparing the application in cancer of pancreas early diagnosis preparation.
The present invention is achieved through the following technical solutions:
The PLGA that the present invention utilizes PEG to modify is as the nano_scale particle carrier of liquid perfluoro bromide octane, and connect can the monoclonal antibody of targeting Early pancreatic carcinoma surface of cell membrane Hedgehog albumen, finally be prepared into the nanoscale ultrasound contrast agents of cancer of pancreas targeting, and have rated its cell in vitro targeting ability.
A first aspect of the present invention, provides a kind of nanoscale ultrasound contrast agents of cancer of pancreas targeting.
The nanoscale ultrasound contrast agents of a kind of cancer of pancreas targeting of the present invention, as shown in Figure 1, is followed successively by from the inside to surface:
Perfluoro bromide octane (PFOB);
The Poly(D,L-lactide-co-glycolide (PLGA) that Polyethylene Glycol (PEG) is modified;
Hedgehog monoclonal antibody;
The Poly(D,L-lactide-co-glycolide (PLGA) specifically modified using Polyethylene Glycol (PEG) is as polymer carrier, parcel perfluoro bromide octane (PFOB), and connect Hedgehog monoclonal antibody on its surface by Polyethylene Glycol (PEG).
Nanoscale ultrasound contrast agents of the present invention, its particle diameter is 100 ~ 500nm, and the nanometer beyond this particle size range is not suitable for, and the too small meeting of particle diameter weakens imaging effect, and particle diameter is crossed conference and reduced contrast agent permeability in the blood vessel, and affects its Targeting Effect.
Hedgehog monoclonal antibody of the present invention, can be commercially available, such as Britain Abcam company etc.
The Poly(D,L-lactide-co-glycolide (PLGA) that the present invention modifies with Polyethylene Glycol (PEG) wraps up perfluoro bromide octane (PFOB), method used is emulsifying volatility process, can see document: Cheng J J, Teply B A, Sherifi, Sung J, Luther G, Gu F X, et al.Formulation of functionalizedPLGA-PEG nanoparticles in vivo targeted drug delivery [J] .Biomaterials, 2007,28:869-876.
A second aspect of the present invention, is to provide the preparation method of the nanoscale ultrasound contrast agents of above-mentioned cancer of pancreas targeting.
The preparation method of the nanoscale ultrasound contrast agents of a kind of cancer of pancreas targeting of the present invention, the method comprises the following steps:
The synthesis of A, PLGA-PEG-NHS
PLGA-COOH with 1-3 (-3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC ﹒ HCl) and N-hydroxyl sulfuration butanimide (NHS) are reacted pre-activate and are become butanimide, then with NH
2-PEG-COOH reaction generates PLGA-PEG-COOH; The PLGA-PEG-COOH of gained reacts with NHS and EDC ﹒ HCl and generates PLGA-PEG-NHS;
The preparation of B, nanoscale ultrasound contrast agents
10 ~ 100mg PLGA-PEG-NHS is dissolved in 1mL dichloromethane solution, add perfluoro bromide octane (PFOB) 5 ~ 50 μ L and 1mg fluorescent dye Coumarin-6 and (add the Targeting Performance that fluorescent material Coumarin-6 can be used for evaluating acoustic contrast agent, therefore can not add), pour into after abundant mixing in the sodium cholate solution of 1%, ice-water bath 400W ultrasonic emulsification 30-100 second, volatilize dichloromethane;
The connection on C, antibody and nanoscale ultrasound contrast agents surface
The nanoscale ultrasound contrast agents that step B is obtained is scattered in PBS solution (pH7.4), adds Hedgehog monoclonal antibody, hybrid reaction, is separated the targeted nano level acoustic contrast agent of the antibody modification obtained through labelling; Lucifuge stirring reaction more than 4 hours (being preferably 6 hours), high speed centrifugation (10000rpm is best) collects nanoscale ultrasound contrast agents, lyophilizing.
The weight ratio of described nanoscale ultrasound contrast agents and Hedgehog monoclonal antibody is good with 50-100:1.
In steps A of the present invention, preferably, the weight ratio of PLGA-COOH and 1-3 (-3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC ﹒ HCl) is 1:8.
Use 8 times amount EDC to activate the carboxyl of PLGA-COOH, strengthen the activity of reacting with NHS.Meanwhile, use the technology of PEG to modify and PLGA nano-carrier can be made comparatively ripe and stable, and non-specific the engulfing that nanoscale ultrasound contrast agents is subject to reticuloendothelial system (RES) can be reduced further.
In step B of the present invention, perfluoro bromide octane (PFOB) is wrapped in the PLGA carrier that PEG modifies, PFOB and PLGA-PEG-NHS in the ultrasonic mixing of ice-water bath, to ensure that macromolecule carrier wraps up the homogeneity of PFOB.Preferably, ice-water bath 400W ultrasonic emulsification 30 seconds, 10 seconds, interval, repeats 3 times.
In step B of the present invention, preferably, PLGA-PEG-NHS 20mg, dichloromethane 1mL perfluoro bromide octane (PFOB) 10 μ L.
In step C of the present invention, preferably, nanoscale ultrasound contrast agents 10mg, Hedgehog monoclonal antibody 200 μ g.
A third aspect of the present invention, the nanoscale ultrasound contrast agents being to provide above-mentioned cancer of pancreas targeting is preparing the application in cancer of pancreas early diagnosis preparation.
Invention further provides the application of described nanoscale ultrasound contrast agents in the diagnosis of cancer of pancreas targeting, described application comprises following content:
Nanoscale ultrasound contrast agents is joined respectively in the human pancreas cancer SW1990 cell strain of Hedgehog high expressed and the human pancreas cancer CFPAC-1 cell strain of the low expression of Hedgehog, observe and detect the picked-up situation of cell.Nanoscale ultrasound contrast agents described above and cell strain hatch 1 hour at 37 DEG C, and rational incubation time ensure that contrast agent and Cell binding are incorporated to born of the same parents.Nanometer acoustic contrast agent provided by the invention joins in the SW1990 cell strain with Hedgehog monoclonal antibody preincubate, to prove the ability of targeted nano level acoustic contrast agent targeting in conjunction with tumor cell further.
Experimental result finds that the SW1990 cell of Hedgehog antigen high expressed is many to the intake of targeted nano level acoustic contrast agent, and green fluorescence distributes relatively more even in cell, and fluorescence intensity is apparently higher than the matched group of non-targeted nanoscale ultrasound contrast agents.The effect of targeted nano level acoustic contrast agent is after 1 hour, and the average fluorescent strength of SW1990 cell is 1463.9, but not the SW1990 cell average fluorescent strength of targeted nano level acoustic contrast agent effect only has 352.6, is only the former 1/4th.
Enter in the cancer of pancreas SW100 cell of Hedgehog high expressed to visible nanometer acoustic contrast agent energy targeting, illustrate that the PLGA nanoscale ultrasound contrast agents of parcel PFOB is suitable as the nanoscale ultrasound contrast agents of cancer of pancreas targeting.
Beneficial effect of the present invention:
The PLGA Biodegradable material that nanoscale ultrasound contrast agents of the present invention adopts PEG to modify is nano-carrier, the nanoscale ultrasound contrast agents good stability formed, size is even, can meet the requirement penetrating tumor vessel wall, reaches the object of being detained at tumor locus and accumulating.Liquid fluorocarbon is different from the ultrasound microbubble contrast agent of inborn reflex and backscattering feature, and its performance producing ultrasonic enhancing development is based on targeting is assembled, thus accuracy is differentiated in raising greatly.Perfluoro bromide octane (PFOB) is the representative compound in liquid fluorocarbon contrast agent.Hedgehog albumen high expressed in cancer of pancreas, and high expressed state exists in early days at neoplastic lesion, and in normal person, the distribution of Hedgehog albumen is limited, therefore targeting Hedgehog is very meaningful for cancer of pancreas early lesion.
Preparation method of the present invention is simple to operate, and reaction reagent is very little with the product toxicity obtained, and can not produce pollute environment, reaction condition is gentle, the nanoscale ultrasound contrast agents simple purification obtained after reaction, with low cost, be beneficial to large-scale promotion in investigation and application field.A first aspect of the present invention, is to provide a kind of nanoscale ultrasound contrast agents of cancer of pancreas targeting.
Accompanying drawing illustrates:
Fig. 1 is the model of nanoscale ultrasound contrast agents of the present invention.
Fig. 2 is the PEG-PLGA-NHS's that synthesizes of the present invention
1hNMR collection of illustrative plates.
Fig. 3 is the nanoscale ultrasound contrast agents transmission electron microscope picture prepared under the different prescription of the present invention; Wherein A, B, C, D, E, F represent and respectively organize prescription accordingly.
Fig. 4 is the particle size distribution rectangular histogram of targeted nano level acoustic contrast agent prepared by the present invention.
Fig. 5 is the Potential distribution rectangular histogram of targeted nano level acoustic contrast agent prepared by the present invention.
Fig. 6 is the fluorine qualitative characterization (D of nanoscale ultrasound contrast agents of monoclonal antibody connection prepared by the present invention
2o does interior mark).
Fig. 7 is the external degree release profiles of targeted nano level acoustic contrast agent prepared by the present invention.
Fig. 8 is the picked-up situation of cell under the targeted nano level acoustic contrast agent fluorescence microscope prepared of the present invention.
Fig. 9 is the picked-up situation of cell under the targeted nano level acoustic contrast agent flow cytomery prepared of the present invention; Wherein upper row is SW1990 cell, and lower row be CFPAC-1 cell, the average fluorescent strength of what block diagram showed is each group of cell.
Detailed description of the invention:
Below in conjunction with the drawings and specific embodiments, the invention will be further described.Should be understood that following examples only for illustration of the present invention but not for limiting scope of the present invention.
The synthesis of embodiment 1:PLGA-PEG-NHS
2000mg PLGA-COOH (purchased from American LAKESHORE company) is dissolved in 5mL dichloromethane, 50mg N-hydroxy-succinamide (NHS) (purchased from Chemical Reagent Co., Ltd., Sinopharm Group), 100mg 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl) (available from Sigma) is added in solution, lucifuge stirring reaction 12 hours, nitrogen dries up dichloromethane, add ether sedimentation and wash, vacuum drying obtains PLGA-NHS.
The PLGA-NHS 1000mg taking synthesis is dissolved in 2mL chloroform, adds 300mgNH
2-PEG-COOH (purchased from Jiaxing Bo Mei Bioisystech Co., Ltd), to react after 12 hours precipitation in methanol and nitrogen dries up and obtains PLGA-PEG-COOH.
Be dissolved in by 500mg PLGA-PEG-COOH in 2mL dichloromethane, add 15mg NHS and 25mg EDCHCl in solution, fully ether sedimentation washing after reaction, vacuum drying obtains the material PLGA-PEG-NHS preparing nanoscale ultrasound contrast agents.
Synthetic product is dissolved in deuterochloroform (CDCl3), utilizes 300MHz nuclear-magnetism to identify (Fig. 2).
Wherein peak a (δ 4.5 ~ δ 4.8), b (δ 5.2), the characteristic peak that c (δ 1.5) is PLGA, peak d is the characteristic peak of PEG (δ 3.6), the characteristic peak that peak e (δ 4.1 ~ δ 4.3) is NHS.
Nuclear magnetic resonance map shows that synthesized material is for preparing the target product PLGA-PEG-NHS needed for nanoparticle.
Embodiment 2: the PLGA nanoscale ultrasound contrast agents preparation prescription screening of parcel PFOB
A certain amount of PLGA and 10 μ L PFOB (available from Sigma) is dissolved in 1mL dichloromethane, then mixes with the sodium cholate solution of 5mL 1%.Under ice bath, ultrasonic 30s stops 10s, and power is 400W, and repeatedly after three times, at room temperature opening stirs and vapors away dichloromethane in 4 hours, and table 1 is the composition of different material.
Utilize current potential particle size analyzer determination particle diameter and current potential (table 2), transmission electron microscope observing form (Fig. 3), wherein from particle diameter and potential measurement result, A group prescription is optimum prescription.
The proportion of composing of table 1 different material
The particle diameter of the nanoscale ultrasound contrast agents of the different formula preparation of table 2 and potential measurement result
| Particle diameter/nm | Polydispersity coefficient | Current potential/mV | |
| A | 198 | 0.11 | -36.8 |
| B | 241 | 0.17 | -40.2 |
| C | 355 | 0.19 | -42.6 |
| D | 454 | 0.34 | -35.9 |
| E | 630 | 0.42 | -44.1 |
| F | 726 | 0.46 | -36.1 |
The preparation of the nanoscale ultrasound contrast agents that embodiment 3:Hedgehog monoclonal antibody connects
Nanoscale ultrasound contrast agents 10mg prepared by the prescription A under Example 2 is dissolved in 5mL PBS solution, add 200 μ g Hedgehog monoclonal antibodies (purchased from Abcam company of Britain), lucifuge stirring reaction 6 hours, centrifugal rear washing precipitation, namely lyophilizing obtains the nanoscale ultrasound contrast agents of cancer of pancreas targeting.Adopt
19f-NMR characterizes, and fluorine qualitative characterization sees Fig. 6.
F content (μ g/g) 161.1, calculates in nanoparticle, and PFOB content is 2.4mg.
The particle size distribution of the nanoscale ultrasound contrast agents of preparation is shown in Fig. 4, and zeta Potential distribution is shown in Fig. 5.Current potential particle size analyzer measures targeted nano contrast agent within the scope of 95 ~ 370nm in normal distribution (Fig. 4), mean diameter is 198.9nm, polydispersity coefficient PDI is 0.26, this size can make full use of the EPR effect of tumor, and nano-contrast agent is detained and accumulation at tumor locus; The surperficial Zeta potential of targeted nano contrast agent is-31.8mV (Fig. 5), the absolute value of Zeta potential is more than 30mV, and the net charge on nanoparticle surface is enough to allow between microgranule and produces larger electrostatic repulsion, and nanoparticle stability is improved, reduce clustering phenomena, keep size even.
Embodiment 4: the research of nanoscale ultrasound contrast agents drug loading and envelop rate
Get nanoscale ultrasound contrast agents 10mg, add 10mL methanol and destroy nanoscale ultrasound contrast agents in ultrasonic 3 minutes, high speed centrifugation is after 5 minutes, getting supernatant utilizes gas chromatography-mass spectrography (GC-MS) method to measure the content of PFOB in nanoscale ultrasound contrast agents, calculates drug loading and envelop rate.GC conditions is: DB-5MS UI chromatographic column (30m × 0.25mm × 0.25 μm), initial column temperature 60 DEG C, 180 DEG C are risen to the speed of 8 DEG C/min, and then rise to 250 DEG C with the speed of 20 DEG C/min, keep 2min, to drain sample, injector temperature 250 DEG C, detector temperature 280 DEG C, carrier gas is helium, sample size 1 μ L.Mass Spectrometry Conditions is: EI ion source, ion source temperature 230 DEG C, flow 1.0mL/min.After sample introduction, the deferred run time of solvent is 3.5min.The envelop rate of nanoscale ultrasound contrast agents is 71.8% ± 1.2% (n=3), and drug loading is 16.1% ± 0.7% (n=3).
Embodiment 5: the mensuration of nanoscale ultrasound contrast agents extracorporeal releasing characteristic
Get 20mg nanoscale ultrasound contrast agents, add PBS solution and be diluted to 2mL, to load in bag filter (MWCO 3500) and seal bag mouth, bag filter puts into constant temperature (37 DEG C) PBS solution of 100mL subsequently, is to carry out drug release determination under the condition of 100 turns per minute in stir speed (S.S.).Drew respectively at 0.5,1,2,3,6,9,12,18,24,36,48 hour the burst size that 1mL PBS measures PFOB, and add 1mLPBS.
As seen from Figure 7, PFOB release in nanoscale ultrasound contrast agents can be divided into 3 stages, first stage release ratio is very fast, within first 3 hours, have one to dash forward to release, burst size accounts for 33.2% of total dose, this is that the PFOB on nanoscale ultrasound contrast agents top layer departs from the quick release caused from nanoscale ultrasound contrast agents, a state steadily discharged is entered after 3 hours, release liquid progressively enters into nanoscale ultrasound contrast agents inside, cause PFOB to top layer diffusion thus enter in release liquid, within first 24 hours, add up to release 71.6%, after 24 hours, medicine diffuses into a poised state, PFOB release in nanoscale ultrasound contrast agents slows down.First 48 hours PFOB add up to release 81.1%.
Embodiment 6: the targeting Journal of Sex Research of targeted nano acoustic contrast agent
The human pancreas cancer SW1990 cell strain of Hedgehog high expressed and the human pancreas cancer CFPAC-1 cell strain (equal purchased from American ATCC company) of the low expression of Hedgehog is adopted to evaluate the targeting carrying PFOB nanoscale ultrasound contrast agents, cell culture fluid is the DMEM culture fluid adding 10%FBS, and culture environment is 37 DEG C, 5%CO
2incubator.Take the logarithm trophophase cell according to every hole 2 × 10
5individual density is inoculated in 6 orifice plates, cultivates 24h and makes cell attachment.Every hole adds 100 μ L nanoscale ultrasound contrast agents solution (500 μ g/mL) afterwards, 37 DEG C hatch 1h after, PBS rinse 3 times, utilizes the cellular uptake situation of fluorescence microscope nanoscale ultrasound contrast agents.Utilize pancreatin by cell dissociation afterwards, centrifugal resuspended to 500 μ L, utilize flow cytometric analysis cell to the picked-up of nanoscale ultrasound contrast agents.
For evaluating targeting Hedgehog monoclonal antibody further nanoscale ultrasound contrast agents entered to the effect of born of the same parents, cell adds excessive Hedgehog monoclonal antibody (10 μ g) in advance and hatches 30min, close Hedgehog protein receptor, then every hole adds 100 μ L nanoscale ultrasound contrast agents solution (500 μ g/mL), 37 DEG C hatch 1h after, carry out observing and detecting respectively by fluorescence microscope and flow cytometer.
Utilize fluorescence microscope pancreatic cancer cell to the picked-up (Fig. 8) of targeted nano level acoustic contrast agent, can find that the intake of the SW1990 cell of Hedgehog antigen high expressed to targeted nano level acoustic contrast agent is many, green fluorescence distributes relatively more even in cell, and fluorescence intensity is apparently higher than the matched group of non-targeted nanoscale ultrasound contrast agents; And the intake of CFPAC-1 cell to targeted nano level acoustic contrast agent not expressing Hedgehog antigen is not significantly increased, illustrate that the nanoscale ultrasound contrast agents that Hedgehog monoclonal antibody is modified has targeting to the pancreatic cancer cell of expressing Hedgehog antigen.After utilizing the Hedgehog antigen on excessive Hedgehog monoclonal antibody closing cell surface, the intake of SW1990 cell to targeted nano level acoustic contrast agent significantly declines, show at SW1990 cell in the capture process of targeted nano level acoustic contrast agent, the endocytic pathway of Hedgehog Ag-Ab mediation has played important function.
Flow cytometer is utilized to find (Fig. 9) to the quantitative study that nanoscale ultrasound contrast agents absorbs, the effect of targeted nano level acoustic contrast agent is after 1 hour, the average fluorescent strength of SW1990 cell is 1463.9, but not the SW1990 cell average fluorescent strength of targeted nano level acoustic contrast agent effect only has 352.6, be only the former 1/4th, and after utilizing antibody to close Hedgehog antigen, the cell average fluorescent strength of targeted nano level acoustic contrast agent effect drops to 414.1, declines clearly.And whether the average fluorescent strength of the CFPAC-1 cell of the low expression of Hedgehog antigen and nanoscale ultrasound contrast agents surface are connected antibody associates not quite, the cell average fluorescent strength of targeting and non-targeted nanoscale ultrasound contrast agents group is respectively 389.7 and 205.6.
Comprehensive above each embodiment, enters in the cancer of pancreas SW100 cell of Hedgehog high expressed to visible nanometer acoustic contrast agent energy targeting, illustrates that the PLGA nanoscale ultrasound contrast agents of parcel PFOB is suitable as the nanoscale ultrasound contrast agents of cancer of pancreas targeting.
Below the preferred embodiment of the invention is illustrated, but the invention is not limited to described embodiment, those of ordinary skill in the art also can make all equivalent modification or replacement under the prerequisite without prejudice to the invention spirit, and these equivalent modification or replacement are all included in the application's claim limited range.
Claims (9)
1. a nanoscale ultrasound contrast agents for cancer of pancreas targeting, is characterized in that, described nanoscale ultrasound contrast agents composition is followed successively by from the inside to surface:
Perfluoro bromide octane;
Polyethyleneglycol modified Poly(D,L-lactide-co-glycolide;
Hedgehog monoclonal antibody;
Described polyethyleneglycol modified Poly(D,L-lactide-co-glycolide, as polymer carrier, wraps up perfluoro bromide octane, and connects Hedgehog monoclonal antibody on its surface by Polyethylene Glycol;
The particle diameter of described nanoscale ultrasound contrast agents is 100 ~ 500nm.
2. the nanoscale ultrasound contrast agents of a kind of cancer of pancreas targeting according to claim 1, is characterized in that, described polyethyleneglycol modified Poly(D,L-lactide-co-glycolide, as polymer carrier, wraps up perfluoro bromide octane by emulsifying volatility process.
3. a preparation method for the nanoscale ultrasound contrast agents of cancer of pancreas targeting as claimed in claim 1, it is characterized in that, the method comprises the following steps:
The synthesis of A, PLGA-PEG-NHS
PLGA-COOH with 1-3 (-3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxyl sulfuration butanimide reaction pre-activate become butanimide, then with NH
2-PEG-COOH reaction generates PLGA-PEG-COOH; The PLGA-PEG-COOH of gained reacts with NHS and EDC ﹒ HCl and generates PLGA-PEG-NHS;
The preparation of B, nanoscale ultrasound contrast agents
10 ~ 100mg PLGA-PEG-NHS is dissolved in 1mL dichloromethane solution, adds perfluoro bromide octane 5 ~ 50 μ L, fully pour in the sodium cholate solution of 1% after mixing, ice-water bath 400W ultrasonic emulsification 30-100 second, volatilize dichloromethane;
The connection on C, antibody and nanoscale ultrasound contrast agents surface
The nanoscale ultrasound contrast agents obtained by step B is scattered in the PBS solution of pH7.4, adds Hedgehog monoclonal antibody, hybrid reaction, is separated the targeted nano level acoustic contrast agent of the antibody modification obtained through labelling; Lucifuge stirring reaction more than 4 hours, high speed centrifugation collects nanoscale ultrasound contrast agents, lyophilizing.
4. the preparation method of the nanoscale ultrasound contrast agents of cancer of pancreas targeting according to claim 3, is characterized in that, in steps A, the weight ratio of PLGA-COOH and 1-3 (-3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride is 1:8.
5. the preparation method of the nanoscale ultrasound contrast agents of cancer of pancreas targeting according to claim 3, is characterized in that, in step B, ice-water bath 400W ultrasonic emulsification 30 seconds, 10 seconds, interval, repeats 3 times.
6. the preparation method of the nanoscale ultrasound contrast agents of cancer of pancreas targeting according to claim 3, is characterized in that, in step B, and PLGA-PEG-NHS 20mg, dichloromethane 1mL, perfluoro bromide octane 10 μ L.
7. the preparation method of the nanoscale ultrasound contrast agents of cancer of pancreas targeting according to claim 3, is characterized in that, in step C, the weight ratio of described nanoscale ultrasound contrast agents and Hedgehog monoclonal antibody is 50-100:1.
8. the preparation method of the nanoscale ultrasound contrast agents of cancer of pancreas targeting according to claim 3, is characterized in that, in step C, and nanoscale ultrasound contrast agents 10mg, Hedgehog monoclonal antibody 200 μ g.
9. the nanoscale ultrasound contrast agents of a cancer of pancreas targeting as claimed in claim 1 is preparing the application in cancer of pancreas early diagnosis preparation.
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| CN107362374A (en) * | 2017-09-08 | 2017-11-21 | 中南大学湘雅三医院 | Carboxylated polylactic-co-glycolic acid wraps up the preparation method of PFOB nanometer acoustic contrast agents |
| CN110152024A (en) * | 2019-06-05 | 2019-08-23 | 深圳市人民医院 | A kind of ultrasound is with magnetic resonance bimodal targeted nano granule contrast agent and preparation method thereof |
| CN111053923A (en) * | 2019-03-27 | 2020-04-24 | 山西省人民医院 | Ultrasonic contrast agent for targeting tumor |
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| CN106581698A (en) * | 2016-12-21 | 2017-04-26 | 中国人民解放军总医院 | Preparation method for ultrasonic fluorescence bimodal nano-probe for recognizing unstable plaque of atherosclerosis |
| CN107362374A (en) * | 2017-09-08 | 2017-11-21 | 中南大学湘雅三医院 | Carboxylated polylactic-co-glycolic acid wraps up the preparation method of PFOB nanometer acoustic contrast agents |
| CN111053923A (en) * | 2019-03-27 | 2020-04-24 | 山西省人民医院 | Ultrasonic contrast agent for targeting tumor |
| CN111053923B (en) * | 2019-03-27 | 2022-03-08 | 山西省人民医院 | Ultrasonic contrast agent for targeting tumor |
| CN110152024A (en) * | 2019-06-05 | 2019-08-23 | 深圳市人民医院 | A kind of ultrasound is with magnetic resonance bimodal targeted nano granule contrast agent and preparation method thereof |
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