CN102008736B - Target cyclopeptide modified liposome microbubble and preparation method thereof - Google Patents
Target cyclopeptide modified liposome microbubble and preparation method thereof Download PDFInfo
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Abstract
The invention provides a target cyclopeptide modified liposome microbubble and a preparation method thereof. The target cyclopeptide modified liposome microbubble comprises a liposome microbubble with streptavidin, and is characterized in that the liposome microbubble is connected with biotin-polyethylene glycol-target cyclopeptide which can be combined with a receptor on the surface of an endothelial cell of a new vessel in hepatofibrosis or cirrhosis through the affinity effect of the streptavidin and biotin. The invention also provides the method for preparing the target cyclopeptide modified liposome microbubble. The target cyclopeptide modified liposome microbubble applied to ultrasonic contrast has the advantage of higher sensitivity.
Description
Technical field
The present invention relates to liposome microvesicle of a kind of targeting cyclic peptide modification and preparation method thereof, assess early stage hepatic fibrosis as acoustic contrast agent and the ultrasonoscopy of integrin alpha v beta 3 receptors bind.
Background technology
So far, the liver biopsy is still unique " goldstandard " of assessment hepatic fibrosis.Yet the liver biopsy has wound, can cause fatal complication, therefore can not generally be accepted by the patient, and this has caused the delay of chronic hepatitis patients in diagnosis and treatment greatly; Simultaneously, in the patient of non-evident sympton, be difficult to repeat the liver biopsy, this just can't follow up a case by regular visits to the variation of the chronic hepatitis patients state of an illness exactly; In addition, the discordance of sample error of sampling and pathological observation has all influenced the accuracy of liver biopsy.Therefore, need a kind of method of accurate non-invasive to diagnose and assess hepatic fibrosis.At present, the appraisal procedure of hepatic fibrosis non-invasive has: 1 fibrosis serum markers.Organ specificity is poor, is subject to the interference of inflammation and organism metabolism, the standardization difficulty, the more reflection inflammation of combined index are difficult by stages to fibrosis; 2 biochemical indicators.Need multinomial combination (like Fibrotest).Because the extremely strong reserve function of liver only when the severe hepatic fibrosis, change obviously, and the biochemical indicator influence factor is many, explains difficulty, the accuracy of the hepatic fibrosis assessment of the different causes of disease, different times is different; 3 conventional images.Present conventional B ultrasonic, CT, MRI are difficult to reflect that hepatic fibrosis changes in early days, and assessment is limited to liver cirrhosis and complication more; 4 developing image technologies.In recent years release ultrasound detection low-frequency elastic ripple instantaneous elasticity monitor (Fibroscan) and the utilization MRI diffusion-weighted imaging (DWI),
31The phosphorus spectroscopic imaging (
31MRS) and new technique such as elastogram variation, assess the hepatic fibrosis degree, be superior to other noninvasive method, have certain application prospect through measuring the hepatic tissue consistency and elasticity.Yet also there is certain limitation in recent discovering, influence the detection accuracy to hepatic fibrosis such as Fibroscan to a great extent like the content of stomach wall fat, and these methods has difficulties also in the diagnosis to early stage hepatic fibrosis.
In sum; Current diagnosis all fails better to reflect activating cell phenotype and number change equimolecular pathological change in the chronic hepatic injury repair process with the method for assessment hepatic fibrosis; And there is discordance at aspects such as reliability, sensitivity, operability, the still difficult requirement that reaches clinical to the assessment of hepatic fibrosis early diagnosis and therapy effect Real-time and Dynamic.Can not make correct assessment to hepatic fibrosis is that clinical " bottleneck " moved towards in restricting foundation research all the time, also is the key link that can not develop anti-fibrosis medicine and clinical trial.
Summary of the invention
The purpose of this invention is to provide liposome microvesicle of a kind of sensitivity that can improve ultrasonic development and preparation method thereof.
In order to achieve the above object; The invention provides the liposome microvesicle that a kind of targeting cyclic peptide is modified; Comprise the liposome microvesicle that has streptavidin; It is characterized in that said liposome microvesicle is connected the targeting cyclic peptide of biotin-Polyethylene Glycol-can combine with the receptor on the new vessels endothelial cell surface in hepatic fibrosis or the liver cirrhosis with the affinity interaction of biotin through streptavidin.The mixture of parcel nitrogen and perfluorocarbon plays the ultrasonic development effect in the liposome microvesicle.
Receptor in described hepatic fibrosis or the liver cirrhosis on the new vessels endothelial cell surface is at least a receptor that under pathological state, raises, like the integrin alpha v beta 3 receptor.
Described targeting cyclic peptide is ring (arginine-glycine-aspartic acid-tyrosine-lysine), i.e. c (RGDyK), and it can combine hepatic stellate cell by specificity, thereby reaches the specificity liver fibrosis diagnosis and improve the purpose of diagnostic sensitivity.Arginine-glycine-aspartic acid in the cyclic peptide (RGD) sequence is the multiple integrin receptor binding site of new vessels endotheliocyte.Wherein, y represents the dextrorotation tyrosine residue, and K represents lysine residue and do not influence with receptor targeted and combines.
The present invention also provides the method for preparing of the liposome microvesicle of above-mentioned targeting cyclic peptide modification, it is characterized in that concrete steps are:
The first step: with mol ratio is that targeting cyclic peptide and the biotin-Polyethylene Glycol-N-hydroxy-succinamide ester of 1.1 ~ 1.3:1 is dissolved in the phosphate buffer of pH=8 jointly; Stirring at room 1-5 hour; Filter; Obtain targeting cyclic peptide-Polyethylene Glycol-biotin bullion, use the preparation HPLC purification, lyophilization gets pure article;
Second step: pure article of targeting cyclic peptide-Polyethylene Glycol-biotin that the first step obtained on ice and the liposome microvesicle that has streptavidin are dissolved in the normal saline respectively; Above-mentioned two kinds of normal saline solutions are mixed, obtain the liposome microvesicle that the targeting cyclic peptide is modified.
The method for preparing of described c (RGDyK) is following: utilize the fluorenylmethyloxycarbonyl solid-phase peptide synthesis; With O-(IH-BTA-1-yl)-N; N; N'; N'-tetramethyl isourea phosphorus hexafluoride and diisopropylethylamine are condensing agent, and (Fmoc-Gly-2-Cl-Trt resin) inserts arginine (R), lysine (K), d-tyrosine (y) and the aspartic acid (D) of side chain protected on fluorenylmethyloxycarbonyl-glycine-dichloro trityl resin successively, after 1% trifluoroacetic acid (TFA) cuts, obtain wire peptide chain D (otBu)-Y (tBu)-K (Boc)-R (the Pbf)-G of side chain protected.Again under the catalysis of DIEA, carry out cyclisation and get with hexafluorophosphoric acid BTA-1-base-oxygen base tripyrrole alkyl phosphorus (PyBOP).
Principle of the present invention is following:
1. (arginine-glycine-asparagic acid, RGD) series is to integrate the plain the most common recognition site that combines aglucon to arginine-glycine-aspartic acid.Integrin alpha v beta 3 can be discerned the RGD part of special conformation among the ECM.Synthetic contains RGD peptide chain high flux screening and finds; Arginine-glycine-aspartic acid acid-(dextrorotation) tyrosine-lysine (c (RGDyK)), arginine-glycine-aspartic acid acid-(dextrorotation) phenylalanine-lysine pentapeptide molecules such as (RGDfK) of head and the tail cyclization also can with the combination of integrin alpha v beta 3 receptor-specific, active amino on the lysine side-chain in the pentapeptide is carried out small numerator modifiedly can't combining activity with aglucon by the appreciable impact receptor.
In our early-stage Study, find that integrin alpha v beta 3 is expressed obviously among the activation HSCs to raise that c (RGDyK) polypeptide in early days can be as a kind of potential tracer in the diagnosis of hepatic fibrosis.This part result of study has been delivered the SCI article: Biochemical characterization of the binding of cyclic RGDyK to hepatic stellate cells. Xiao-wei Huang; Ji-Yao Wang; Et al. Biochemical Pharmacology. 2010; (80:136-143. IF=4.8,2008)
In addition; We also find at thioacetamide (thioacetamide; TAA) and the common bile duct ligation (bile duct ligation, BDL) in inductive two kinds of rat liver fibrosis models, integrin alpha v beta 3 and a-smooth muscle actin (a-smooth muscle actin; It is closely related that α-SMA) is expressed on degree and the position, and the Fibrotic development trend of its regulating liver-QI is consistent.And the interior research of the preliminary body that has carried out the SPECT video picture.
2. present, external existing much through the integrin alpha v beta 3 expression of receptor being carried out the molecular imaging research of video picture with the contrast medium of RGD cyclic peptide labelling.Groundwork concentrates on the aspects such as bone reconstruction that tumor neogenetic blood vessels, blood vessel injury are reinvented, osteoclast is participated in.Though the conventional ultrasonic diagnosing liver fibrosis in early days that is difficult to; But the application of ultrasonic microbubble has greatly improved acoustic picture; And the application of targeted ultrasound microvesicle can make ultrasonic image be extended to the molecular image field; Radiography quantitative analysis software can be gathered the sequence dynamic arthrography image in the survey region, analyzes the variation of the amount of its interior each pixel and contrast agent bubble echo, thereby can obtain to reflect the blood flow parameter of cell function variation.External expression and the intervention back of this technology understanding integrin alpha v beta 3 when the experimental tumor angiogenesis of once using changes.And in hepatic fibrosis, do not see relevant research report at present.
Advantage of the present invention is:
1. the advantage of molecular imaging technology is that it can quantize living body biological process, avoids sampling error and remedies the defective that histologic analysis can not provide function information from room and time; In addition, because the molecular imaging means can carry out dynamic studies to the developed by molecule pattern of same animal, so can significantly reduce the demand of animal and increase the credibility of result of study.
2. ultrasound imaging techniques is superior to other imaging patterns aspect some.For example, ultra sonic imaging is moment and real-time, and the deadline of other most of image forming programs usually will be with minute calculating.Different with nuclear medicine and CT technology, ultra sonic imaging need not to use ionizing radiation.Although nmr imaging technique (MRI) can provide and the similar spatial resolution of micro-ultrasonic system (Vevo770), carrying out NMR-imaging for the motion sample of breathing or heart beating causes is difficulty and consuming time.Up-to-date high frequency micro-ultrasonic imaging technique (Vevo770) can reach the spatial resolution that is close with NMR-imaging; And can be implemented in that non-invasive ground carries out real-time anatomy on the identical platform, hemodynamics and molecular data collection and quantitative analysis research.The miniature ultrasonic imaging can be used on the various material, and requires very low to imaging theory and experience.
3. ultra sonic imaging contrast agent application the scattering properties of microbubble.The most frequently used contrast agent is the gas compressed microparticles that is called as microbubble (microbubbles).Because the impedance contrast at the critical interface of gas/liquid is higher, adds inherent interaction, so acoustic contrast agent is easy to cause than blood or the bigger ultrasound wave backscatter of its hetero-organization.Ultrasonic and high frequency is micro-to routine ultrasonicly all is suitable for for peplos bubble contrast agent microparticles.These contrast agent shells are very thin, and shell contains a small amount of bio-compatible gas.Joint through target molecule part contrast agent shell can be easy to realize the locking of contrast agent to molecular target.Current technology allows multiple potential target molecule part to participate in this conjugation, comprises monoclonal antibody, polypeptide, glycoprotein, and nucleic acid.Microbubble is as a kind of acoustic contrast agent, and its diameter is usually between the 0.5-5 micron, and this is the space of blood vessel under the generic condition just.This feasible detection to the internal blood vessel molecular targets becomes possibility, but the imaging of molecule can't reach in the pair cell.Microbubble can be broken up by hyperacoustic high-voltage pulse.This process of quickly and easily the contrast agent bubble being destroyed makes promptly to be removed these contrast agent in blood, and the contrast agent and the distinguishing of circulation contrast agent of binding molecule target become possibility.Compare the micro-ultrasonic expression that is implemented in the some molecular markers of fast detecting in the single imaging process with the image documentation equipment of other kinds.
4. among the present invention, we at first c (RGDyK) are carried out PEG and Biotin modifies, and are connected the liposome microvesicle surface that has the strepavidin coated shell to Biotin-PEG-c (RGDyK) then.The pharmacokinetics of the PEG of this extension link not only improvement ultrasonic microbubble prolonged the targeted microbubble circulation time, also reduced liposome microvesicle sterically hindered to the bind receptor ability of bonded c (RGDyK) simultaneously as far as possible.
Description of drawings
Fig. 1 is the targeted ultrasound contrast schematic flow sheet;
Fig. 2 is that fibrosis rat liver targeted ultrasound microvesicle strengthens the representative striograph of radiography;
Fig. 3 is average signal strength (MPA) and the time history in the area-of-interest (ROI).
The specific embodiment
Specify the present invention below in conjunction with embodiment.
Embodiment 1
C's (RGDyK) is synthetic:
Utilize fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis; With O-(IH-BTA-1-yl)-N; N; N'; N'-tetramethyl isourea phosphorus hexafluoride (HBTU)/diisopropylethylamine (DIEA) is a condensing agent, and (Fmoc-Gly-2-Cl-Trt resin) inserts arginine (R), lysine (K), d-tyrosine (y) and the aspartic acid (D) of side chain protected on fluorenylmethyloxycarbonyl-glycine-dichloro trityl resin successively, after 1% trifluoroacetic acid (TFA) cuts, obtains wire peptide chain D (otBu)-Y (tBu)-K (Boc)-R (the Pbf)-G of side chain protected.Again under the catalysis of DIEA, carry out cyclisation and get with hexafluorophosphoric acid BTA-1-base-oxygen base tripyrrole alkyl phosphorus (PyBOP).
Detailed process is following: 0.25g Fmoc-Gly-2-Cl-Trt resin (substitution value 1mmol/g) places the polypeptide composite tube, and N is after dinethylformamide (DMF) swelling; 20% piperidines DMF solution is sloughed Fmoc protection base, and (2.2mmol 2,2 to add the arginine reactant liquor; 4; 6,7-pentamethyl Dihydrobenzofuranes-5-sulphonyl-arginine is dissolved in the DMF solution and 1mlDIEA of 4ml 0.5mol/L HBTU), in room temperature vibration 30 minutes; Reaction finishes the back sucking filtration and removes reactant liquor, and with the DMF washing resin.Resin is sloughed Fmoc protection base with 20% piperidines DMF solution again; React tertbutyloxycarbonyl-lysine, the tert-butyl group-tyrosine and tert-butoxy-aspartic acid successively with same method, after last aminoacid inserted resin and sloughs Fmoc protection base, resin with washed with dichloromethane for several times; Add 2ml 1%TFA reaction 2 minutes then; Sucking filtration is collected filtrating, reprocessing 10 times, merging filtrate.After the filtrating rotary evaporation concentrates, add distilled water and separate out deposition, lyophilization gets the wire peptide of side chain protected.The linear peptides of gained is dissolved among the DMF, add 1.1 times to the PyBop of polypeptide amount and DIEA in stirred overnight at room temperature, add elutriation after the reaction and go out deposition, sucking filtration must precipitate.The gained deposition is taken off side chain protected with the 95%TFA reaction; Behind the thin up; With preparation HPLC purification (chromatographic column: C18 300 à, elution process: 5% acetonitrile contains 0.1%TFA aqueous solution to 25% acetonitrile and contains 1 hour gradient elution of 0.1%TFA aqueous solution), lyophilization gets c (RGDyK).
Embodiment 2
(1) c (RGDyK)-PEG-Biotin is synthetic:
The 0.012mol c (RGDyK) that makes is dissolved in 5ml phosphate buffer (pH=8) with 0.01mol biotin-Polyethylene Glycol-N-hydroxy-succinamide ester (biotin-PEG-NHS); In stirring at room 3 hours; Filter, make c (RGDyK)-PEG-Biotin bullion, through preparation HPLC purification (chromatographic column: C18 300 à; Elution process: 35% acetonitrile contains 0.1%TFA aqueous solution to 55% acetonitrile and contains 1 hour gradient elution of 0.1%TFA aqueous solution), lyophilization gets pure article.
(2) preparation of the targeted ultrasound microvesicle of Biotin-PEG-c (RGDyK) modification:
The used liposome microvesicle that has streptavidin is the VS-11675 type product that Canadian Visualsonics company produces.At first connect white lid syringe, inject 500 μ l normal saline to the contrast agent bottle, the above-mentioned liposome microvesicle 10 that has streptavidin is housed in the said contrast agent bottle with green syringe needle
9Individual/ml, abandon needle tubing, let the acupuncture needle remain at a certain point head, balance is pulled out syringe needle after in the several seconds.Soft vibration 10 seconds, room temperature left standstill 5 minutes.20 μ g Biotin-PEG-c (RGDyK) are diluted to 400 μ l final volume with normal saline, are stored in the 1ml Ep pipe.1ml syringe with green syringe needle is transferred to polypeptide in the contrast agent bottle, and final volume is 900 μ l.Soft vibration 15 min, room temperature leaves standstill and obtains the contrast agent that targeted microbubble is modified.
Above-mentioned Biotin-PEG-c (RGDyK) with homotype contrast polypeptide c (RGAyK)-PEG-Biotin replacement, is prepared homotype contrast contrast agent with quadrat method.
The Lycoperdon polymorphum Vitt syringe needle is connected the green lid syringe of preliminary filling 0.7ml normal saline, be used to inject afterflush.Connect the 1ml syringe with green pin, aspirate about 100～150 μ l homotypes contrast contrast agent, it is subsequent use to change the Lycoperdon polymorphum Vitt syringe needle.Same method suction 100～150 μ l target polypeptide contrast agent, it is subsequent use to change the Lycoperdon polymorphum Vitt syringe needle.Aforesaid operations all carries out on ice.
Application examples
It is following that contrast agent that the targeted microbubble that uses embodiment 2 to obtain is modified and homotype contrast contrast agent carry out targeted ultrasound microvesicle radiography:
1, gets into VisualSonics
Vevo770
High-resolution toy ultrasonic system is provided with parameter: RMV 707B 30MHz, Power 50%, RF cycles 1, Frame rate 20, Sequence Destroy Position 30%.
2, with rat anesthesia, body temperature maintains 37 ℃, and it is fixing to lie on the back, and rejects abdominal part Mus hair, selects suitable position to make the liver image in the maximum cross-section, the end of line venous cannulation.
3, carry out targeted ultrasound microvesicle radiography flow process.
4, contrast agent and the homotype contrast contrast agent targeted microbubble modified inject through the rat tail vein intubate.The amount of injecting microvesicle is 100-150 ul (5 * 10
7Microvesicle ≈ 100 ul), use the normal saline flushing of 20 ul then.
5, each rat is all accepted contrast agent and the potion homotype contrast contrast agent that the potion targeted microbubble is modified, front and back 20 minutes at interval with order at random.Immediately with low-yield affirmation microvesicle existing in liver of 10%, stopped subsequently sampling 4 minutes after the injection.
6, through after 4 minutes accumulation phase, image acquisition restarts with 50% energy.Gathered after about 200 frame images, using mid frequency is the microvesicle at the high energy destruction pulse destruction rising peak of 10 MHz.After high energy destroyed pulse, image acquisition restarted with 50% energy immediately, can observe the full again peak value of microvesicle residual in the circulation.
7, interpretation of result:
Adherent microvesicle obtains with destroying-subtract the shadow imaging application.Pixel amplitudes before using the destruction pulse in the liver derives from tissue, adheres to the acoustic responses of the non-adhesion microvesicle in microvesicle and the circulation.Destroy pulse and eliminated the microvesicle the rising peak in, in several seconds after destroying pulse, pixel amplitudes derives from tissue and residual non-adhesion microvesicle in the circulation at additional rising peak again.The mode of spatial distribution through Digital Subtraction that representative adheres to the pixel of microvesicle obtains, and promptly deducts the reference picture that destroys after the pulse the image before destroying pulse.Reference picture is made up of 100 frames (≈ 7 seconds) image, after destroying pulse application, gathers immediately, and removes noise with 3 * 3 low-pass filter in advance.More than of reference pictures ground with similarly compare through filtering contrast image, visual selected come out best with the reference picture dependency does to make that to subtract microbubble signals residual in the shadow image afterwards minimum like this.The best reference picture of dependency is removed before using the destruction pulse with the image of being gathered afterwards with the mode of Digital Subtraction, and the difference of pixel amplitudes is encoded into colored mask and covers on the B ultrasonic image.As shown in Figure 1, be the targeted ultrasound contrast schematic flow sheet.Fig. 2 is that fibrosis rat liver targeted ultrasound microvesicle strengthens the representative striograph of radiography.Fig. 3 is average signal strength (MPA) and the time history in the area-of-interest (ROI).
Claims (3)
1. the liposome microvesicle modified of a targeting cyclic peptide; Comprise the liposome microvesicle that has streptavidin; It is characterized in that said liposome microvesicle is connected the targeting cyclic peptide of biotin-Polyethylene Glycol-can combine with the integrin alpha v beta 3 receptor on the new vessels endothelial cell surface in hepatic fibrosis or the liver cirrhosis with the affinity interaction of biotin through streptavidin; The described targeting cyclic peptide that can combine with the integrin alpha v beta 3 receptor on the new vessels endothelial cell surface in hepatic fibrosis or the liver cirrhosis is ring (arginine-glycine-aspartic acid-tyrosine-lysine); The described liposome microvesicle that has streptavidin is the VS-11675 type product that Canadian Visualsonics company produces.
2. the liposome microvesicle that targeting cyclic peptide as claimed in claim 1 is modified is characterized in that the method for preparing of described ring (arginine-glycine-aspartic acid-tyrosine-lysine) is following:
Utilize the fluorenylmethyloxycarbonyl solid-phase peptide synthesis; With O-(IH-BTA-1-yl)-N; N, N', N'-tetramethyl isourea phosphorus hexafluoride and diisopropylethylamine are condensing agent; On fluorenylmethyloxycarbonyl-glycine-dichloro trityl resin, insert arginine, lysine, d-tyrosine and the aspartic acid of side chain protected successively, after the trifluoroacetic acid cutting, obtain the wire peptide chain of side chain protected; Again under the catalysis of diisopropylethylamine, carry out cyclisation and get with hexafluorophosphoric acid BTA-1-base-oxygen base tripyrrole alkyl phosphorus.
3. the method for preparing of the liposome microvesicle modified of the described targeting cyclic peptide of claim 1 is characterized in that concrete steps are:
The first step: with mol ratio is that targeting cyclic peptide and the biotin-Polyethylene Glycol-N-hydroxy-succinamide ester of 1.1 ~ 1.3:1 is dissolved in the phosphate buffer of pH=8 jointly; Stirring at room 1-5 hour; Filter; Obtain targeting cyclic peptide-Polyethylene Glycol-biotin bullion, use the preparation HPLC purification, lyophilization gets pure article;
Second step: inject on ice in the contrast agent bottle of the VS-11675 type product that 500 μ l normal saline to Canadian Visualsonics company produces; The pure article 20 μ g of targeting cyclic peptide-Polyethylene Glycol-biotin that the first step is obtained are diluted to 400 μ l final volume with normal saline, are transferred in the described contrast agent bottle, obtain the liposome microvesicle that the targeting cyclic peptide is modified.
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| CN102604637B (en) * | 2012-02-10 | 2016-07-06 | 中国科学院福建物质结构研究所 | The preparation method of the rear-earth-doped inorganic fluorescent nano-particle of biotin modification |
| CN106699845A (en) * | 2015-11-12 | 2017-05-24 | 复旦大学 | Stapled-RGD polypeptide, and applications thereof in tumor targeting delivery |
| CN106237342A (en) * | 2016-08-12 | 2016-12-21 | 复旦大学 | A kind of preparation method i.e. joining instant nano target medicine carrier |
| CN109589419B (en) * | 2019-01-17 | 2023-01-31 | 中国人民解放军第四军医大学 | Targeted temperature-controlled polysaccharide-loaded long-circulating liposome-microbubble complex drug delivery system and preparation method thereof |
| CN110124056B (en) * | 2019-05-16 | 2020-08-21 | 中国人民解放军第二军医大学 | Liposome for resisting organ transplant rejection, preparation method and application |
| CN110551180A (en) * | 2019-08-21 | 2019-12-10 | 华南理工大学 | Multifunctional implant with response effect and preparation method and application thereof |
| CN113717246A (en) * | 2021-08-05 | 2021-11-30 | 核欣(苏州)医药科技有限公司 | Preparation method of polypeptide heterodimer |
| CN116370658A (en) * | 2023-03-08 | 2023-07-04 | 中国科学院深圳先进技术研究院 | Tumor-targeted ultrasonic contrast agent and preparation method and application thereof |
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| CN100436477C (en) * | 2005-03-25 | 2008-11-26 | 复旦大学附属中山医院 | Cyclic peptide containing arginine, glycine, asparagicacid-sequence and active target liposome |
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| 宋正杰，王吉耀.《肝星状细胞整合素αvβ3表达及和RGD环肤结合研究》.《第七次全国消化病学学术会议论文汇编》.2007,第834页. * |
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