CN113929740A - Amphiphilic polypeptide and preparation method and application thereof - Google Patents
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- 239000011737 fluorine Substances 0.000 claims abstract description 10
- 238000002604 ultrasonography Methods 0.000 claims description 20
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- UKLDJPRMSDWDSL-UHFFFAOYSA-L [dibutyl(dodecanoyloxy)stannyl] dodecanoate Chemical compound CCCCCCCCCCCC(=O)O[Sn](CCCC)(CCCC)OC(=O)CCCCCCCCCCC UKLDJPRMSDWDSL-UHFFFAOYSA-L 0.000 description 2
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- BDHUTRNYBGWPBL-HNNXBMFYSA-N (2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]-6-(phenylmethoxycarbonylamino)hexanoic acid Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CCCCNC(=O)OCC1=CC=CC=C1 BDHUTRNYBGWPBL-HNNXBMFYSA-N 0.000 description 1
- GJXKBGKAUIMAKK-HNNXBMFYSA-N (2s)-2-[(4-methylphenyl)sulfonylamino]-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound CC1=CC=C(S(=O)(=O)N[C@@H](CCCCNC(=O)OC(C)(C)C)C(O)=O)C=C1 GJXKBGKAUIMAKK-HNNXBMFYSA-N 0.000 description 1
- DYSBKEOCHROEGX-HNNXBMFYSA-N (2s)-6-[(2-methylpropan-2-yl)oxycarbonylamino]-2-(phenylmethoxycarbonylamino)hexanoic acid Chemical compound CC(C)(C)OC(=O)NCCCC[C@@H](C(O)=O)NC(=O)OCC1=CC=CC=C1 DYSBKEOCHROEGX-HNNXBMFYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
- A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Physics & Mathematics (AREA)
- Acoustics & Sound (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Radiology & Medical Imaging (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to an amphiphilic polypeptide and a preparation method and application thereof. Particularly, the invention also provides a copolymerized polypeptide microbubble which has high stability and can enhance ultrasonic contrast in vivo in situ and a method thereof. Specifically, the invention designs and synthesizes a series of fluorine-containing or crosslinkable amphiphilic polypeptide (formulas (A) - (D), wherein each l is independently 2-4; i is 4-11; k is 4-400; n is 1-50; and m is 1-100, the number average molecular weight is 1000-100000), and partial polypeptide can be irreversibly combined with protein to enhance the acoustic performance of the material. The polymer of the invention can well prepare the microvesicle and disperse the microvesicle into aqueous solution, and the microvesicle can be applied to advanced diagnosis and drug delivery, and particularly can effectively generate echo signals during ultrasonic diagnosis, thereby greatly enhancing the signal-to-noise ratio.
Description
Technical Field
The invention belongs to the field of materials and relates to amphiphilic polypeptide as well as a preparation method and application thereof. In particular, the invention relates to the application of amphiphilic polypeptide in preparing copolymerized polypeptide microvesicle with high stability and capability of enhancing ultrasonic contrast in situ in vivo, and further explores the basic properties of microvesicle and potential application in ultrasonic imaging diagnosis.
Background
Ultrasound (US) imaging is widely used and is considered a safe medical tool for disease diagnosis and treatment. The system has the advantages of non-invasiveness, utilization of non-ionizing radiation, relatively low cost, convenience in carrying, convenience in real-time visualization and the like. Micron-sized particles of Microbubbles (MB) formed by encapsulating gas in a biocompatible material have enjoyed great success as Ultrasound Contrast Agents (UCAs) due to their high ultrasound reflectivity and high stability. The contrast of clinical ultrasound is based on changes in acoustic impedance of various tissues, and the contrast of air microbubbles is lower compared to other imaging techniques. Perfluorocarbons (PFCs) are used as gas nuclei for microbubbles due to their insolubility in biological fluids, resulting in reduced volume transfer and extended half-life (Theranostics 2012,2,1185, Reviews: Nanomedicine and Nanobiotechnology 2012,4, 492). While liquid PFC droplets are effective ultrasound contrast agents at high doses, gas phase PFC microbubbles work much better, requiring doses that are 1/30 for grayscale ultrasound imaging (invest. radio.1994, 29, S139), and produce higher image contrast than nonlinear imaging (acad. radio.1998, 5, S63).
Various nanocarriers have been used for PFC-based ultrasound contrast agents, including liposomes, polymeric nanoparticles, proteins, and inorganic nanoparticles. Recently, polypeptids have been developed very rapidly and have found some applications in biomedical polymer fields, such as gene therapy, drug delivery, polypeptid hydrogels, and the like. Therefore, polypeptides having biocompatibility play an important role in biosafety. These materials typically create a functionalized shell around the PFC core to improve stability by lowering surface tension and provide a diffusion barrier. The crosslinked polymer shell provides an option for stabilizing the polymer network and preventing the escape of perfluorocarbons. But DNA-based cross-linking strategies have been used to prevent nonlinear oscillation of microbubbles in response to ultrasound stimuli (adv. mater.2012,24,6010). Meanwhile, Frinking et al proposed a phenomenological model describing the contrast agent Quantison, and studies showed that the shell increased the mechanical stiffness of the contrast agent particles and that the shell viscosity increased the sound absorption (Ultrasound Med. biol.24, 523-533,1998).
In the past, the materials most used in microbubble preparation include phospholipids, polyesters, and polycyanoacrylates. Since the surface tension of the hydrophobic part of these reagents is higher than 30mN/m, encapsulation of the bubbles with a fluorinated polymer having a surface tension as low as 6.7mN/m can greatly improve the stability of the bubbles (Langmuir 1999,15, 4321. Ed.2015,54, 14291. Ed. 14294). It is more stable under the action of ultrasound in water and human blood than commercial third generation ultrasound contrast agents. Research results show that changing the encapsulating material is a feasible method for preparing stable microbubbles and nanobubbles. Future versions of these microbubbles may find use in advanced diagnostics and drug delivery.
Disclosure of Invention
The invention designs and synthesizes a series of amphiphilic polypeptide containing fluorine or crosslinkable, and provides a copolymerized polypeptide microbubble which has high stability and can enhance contrast in situ in vivo, thereby realizing sensitive detection in ultrasonic diagnosis. In particular, the fluorine block in the amphiphilic polypeptide reduces laplace pressure of the microbubbles, greatly enhances the stability of the microbubbles, and further enhances the stability in the case of crosslinking, and some polymer microbubbles can even irreversibly bind to proteins to enhance the acoustic properties of the material. The ultrasound beam causes a compression-tension phenomenon in the medium through which it travels, and the microbubbles exposed to the ultrasound beam likewise undergo a consistent contraction-expansion action. As the acoustic energy is further amplified, the oscillation of the microbubbles becomes nonlinear, so that the resulting echo signals contain not only the frequency at which the microbubbles were initially exposed to ultrasound, the fundamental frequency, but also harmonic frequencies. Since the harmonic signals originate mainly from the microbubbles, the signal-to-noise ratio is greatly enhanced.
In one aspect, the present invention provides a series of novel amphiphilic polypeptides, classified as di-block or tri-block polypeptides, wherein the di-block polypeptides comprise i) a strongly hydrophobic fluoropolypeptide block with low surface tension properties as the inner core and a hydrophilic polyethylene glycol block as the outer group, and the tri-block polypeptides comprise i) a strongly hydrophobic fluoropolypeptide block with low surface tension properties as the inner core and a hydrophilic polyethylene glycol block as the outer group; and ii) a crosslinkable diyne block as a linking block for both.
(A) The amino acid derivative monomers related to the formulas (A), (B), (C) and (D) are shown as the following formulas (a) and (B):
wherein l is 2-4, and i is 4-11.
In one aspect, the invention provides amphiphilic polypeptides of formulae (a), (B), (C), and (D):
wherein each l is independently 2-4; i is 4 to 11; k is 4-400; n is 1 to 50; and m is 1 to 100.
In some embodiments, the amphiphilic polypeptide has a number average molecular weight of 1000-100000.
In another aspect, the present invention provides a method of preparing an amphiphilic polypeptide of formulae (a) and (B), the method comprising:
in another aspect, the present invention provides a method of preparing an amphiphilic polypeptide of formulae (a) and (B), the method comprising: (1) reacting a monomer of formula (a) or monomers of formulae (a) and (B) with a chain initiator of formula (c1) in a solvent, and (2) adding benzyl isocyanate to react to give amphiphilic polypeptides of formulae (A) and (B),
wherein each l is independently 2-4; i is 4 to 11; k is 4-400; n is 1 to 50; and m is 1 to 100.
One specific example is shown in the following scheme:
in some embodiments, the solvent in step (1) is selected from N, N-dimethylacetamide or dimethylsulfoxide.
In some embodiments, the reaction temperature in step (1) is 60-90 ℃.
In some embodiments, the reaction time in step (1) is 3 hours to 2 days.
In some embodiments, the solvent in step (2) is selected from N, N-dimethylacetamide or chloroform.
In some embodiments, the reaction temperature in step (2) is 50-110 ℃.
In some embodiments, the reaction time in step (2) is 1 to 6 hours.
In some embodiments, the chain initiator is a hydrophilic polymer modified at one methoxy end and at the other with a protonated amino group; the method is characterized in that the chain initiator is a hydrophilic polymer with one end being a methoxyl group and the other end being a benzyl protonated amino group modified by carbamate.
In another aspect, the present invention provides a method of preparing an amphiphilic polypeptide of formulae (C) and (D), the method comprising: (1) reacting a monomer of formula (a) or monomers of formulae (a) and (b) with a chain initiator of formula (C2) in a solvent, and (2) adding benzyl isocyanate to react to give amphiphilic polypeptides of formulae (C) and (D),
wherein each l is independently 2-4; i is 4 to 11; k is 4-400; n is 1 to 50; and m is 1 to 100.
One specific example is shown in the following scheme:
in some embodiments, the solvent in step (1) is selected from N, N-dimethylacetamide or dimethylsulfoxide.
In some embodiments, the reaction temperature in step (1) is 60-90 ℃.
In some embodiments, the reaction time in step (1) is 3 hours to 2 days.
In some embodiments, the solvent in step (2) is selected from N, N-dimethylacetamide or chloroform.
In some embodiments, the reaction temperature in step (2) is 50-110 ℃.
In some embodiments, the reaction time in step (2) is 1 to 6 hours.
In some embodiments, the chain initiator is a hydrophilic polymer modified at one end with a maleimide group and at the other end with a protonated amino group; the method is characterized in that one end of the chain initiator is benzyl maleimide modified by carbamate; the other end of the hydrophilic polymer is modified by carbamate to form benzyl protonated amino.
In another aspect, the invention provides polypeptidic microbubbles prepared from an amphiphilic polypeptide of the invention, and methods of preparing the polypeptidic microbubbles. In particular, the microvesicles prepared are size-adjustable, biocompatible polypeptidic microvesicles.
In another aspect, a method of making a polypeptidic microbubble comprises: dissolving amphiphilic polypeptide in organic solvent, evaporating the obtained organic solution to form a film, adding water for hydration, and finally performing ultrasonic treatment on the obtained system in the atmosphere of fluorine-containing gas to obtain the polypeptide microbubble.
In some embodiments, the organic solvent is selected from chloroform, hexafluoroisopropanol or mixtures thereof in any proportion.
In some embodiments, the concentration of amphiphilic polypeptide in the organic solution is between 0.1mg/mL and 100 mg/mL.
In some embodiments, the volume of water added is 0.1 to 100 times the volume of the organic solvent.
In some embodiments, the temperature of hydration is from 25 to 70 ℃, preferably 50 ℃.
In some embodiments, the time of hydration is 2 hours to 3 days, preferably 2 days.
In some embodiments, the fluorine-containing gas is selected from perfluoropropane, perfluorobutane and sulfur hexafluoride, preferably perfluoropropane.
In some embodiments, the sonication time is 10 to 60 seconds.
In some embodiments, the ultrasound amplitude is 40% to 70%.
In some embodiments, the diameter of the microbubbles ranges from 0.5 to 10 μm.
In some embodiments, the microbubbles are used in ultrasound imaging diagnostics.
In another aspect, the invention provides the use of a microbubble of the invention in the manufacture of a medicament for diagnostic ultrasound imaging.
The invention has the advantages of
The previous microbubbles have the disadvantages of poor stability and need to be opened immediately in practical application, and the disadvantages greatly limit the practical application range of the microbubbles. In the past, the materials most used in microbubble preparation include phospholipids, polyesters, and polycyanoacrylates. The surface tension of the hydrophobic part of these reagents is higher than 30mN/m, and the encapsulation of the bubbles with fluorinated polymers having surface tensions as low as 7-12mN/m can greatly improve the stability of the bubbles. On the other hand, the crosslinked polymer shell provides an option for stabilizing the polymer network and preventing the escape of perfluorocarbons. Obviously, this type of microbubble can provide greater environmental tolerance and longer detection time for ultrasound diagnosis. Therefore, the synthesis of the novel polymer microbubble and the application of the novel polymer microbubble in ultrasonic diagnosis and detection have important scientific significance and great practical value.
The copolymerized polypeptide microbubble with high stability and capability of enhancing ultrasonic contrast in vivo in situ realizes sensitive detection in ultrasonic diagnosis. Specifically, a series of amphiphilic polypeptides containing fluorine or capable of being cross-linked are designed and synthesized, wherein the fluorine block reduces the Laplace pressure of the microbubbles, so that the stability of the microbubbles is greatly enhanced, meanwhile, if cross-linking is carried out, the stability can be further enhanced, and partial polymer microbubbles can be even combined with protein irreversibly to enhance the acoustic performance of the material. Future versions of these microbubbles may be applied for advanced diagnostics and drug delivery. In particular, the polymer containing a suitable hydrophobic chain or a suitable hydrophilic glycol chain of the present invention can well prepare microbubbles and disperse them into an aqueous solution, and can effectively generate an echo signal at the time of ultrasonic diagnosis.
Drawings
FIG. 1 shows nuclear magnetic hydrogen spectra (a), nuclear magnetic carbon spectra (b), and high resolution Mass spectra (ESI-Mass) (c) of diyne-containing lysine derivative monomers.
Figure 2 shows nuclear magnetic hydrogen (a), nuclear magnetic carbon (b), nuclear magnetic fluorine (c) and high resolution Mass (ESI-Mass) (d) spectra of diyne-containing lysine derivative monomers.
FIG. 3 shows the structure (a) and nuclear magnetic hydrogen spectrum (b) of the prepared amphiphilic polypeptide.
Fig. 4 shows a TEM photograph of the amphiphilic polymer of the present invention after hydration of the film.
FIG. 5 shows PEG-based45-b-PFOK11(wherein the subscript number "45" indicates the degree of polymerization of PEG and "11" indicates the degree of polymerization of FOK.) the bright field pictures of 2.4 micron microbubbles prepared under a confocal fluorescence microscope.
Figure 6 shows the results of stability of the amphiphilic polypeptide microbubbles of the invention in a skin-vessel angiography.
FIG. 7 shows amphiphilic Polypeptides MI-PEG of the invention45-PFOK11Rabbit in vivo ultrasound imaging of microbubbles.
Detailed Description
The present invention is further illustrated by the following specific examples. The raw materials used in the present invention are illustrated below:
N-Boc-N' -Cbz-L-lysine was purchased from Bibei medicine and used as such. 10, 12-Pentagon is purchased from Alfa Aesar. Albumin from bovine serum was purchased from alatin and used as received. 2-mercaptobenzothiazole, phenyl chloroformate, tert- butyl 2,2, 2-trichloroacetimidate, 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, trifluoroacetic acid, triethylsilane from AnkanjiSchool, and used as received. Pentadecafluorooctanoic acid was purchased from Jiuding, and used as received. Perfluoropropane was purchased from fluorinating technology and used as received. Sodium carbonate (Na)2CO3) Dibutyl tin Dilaurate (DBTL), ethyl acetate (EtOAc), Tetrahydrofuran (THF), methanol, diethyl ether, N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), toluene were purchased from national pharmaceutical chemicals ltd and used as received. The water was Deionized (DI) with Milli-Q SP reagent water system (Millipore) to achieve a resistivity of 18.4M Ω cm. Commercial phospholipid-encapsulated sulfur hexafluoride microbubble samples were obtained from the first subsidiary Hospital of the university of scientific and technology, China, under the trade name SonoVueTM. Unless otherwise indicated, all other reagents were purchased from national pharmaceutical group chemical agents, ltd, and used as received.
Examples
The invention is further illustrated in the following examples, in which R ═ CH is used3Polyethylene glycol (PEG) has a molecular weight of 2000, and k ═ 45 is taken as an example, which is only for better understanding of the present invention and is not intended to limit the scope of the present invention.
Example 1
In a first step, the following lysine derivative 1 (wherein l ═ 4) was prepared:
it is characterized in that: the pendant group of na-t-butoxycarbonyl-lysine t-butyl ester reacts with an alkyl chain containing a diyne group.
The preparation method comprises the following steps: n α -tert-Butoxycarbonyl-lysine tert-butyl ester (3.7g, 12.2mmol) and 10, 12-pentahexadienoic acid (5.5g, 14.7mmol) were dissolved in 40mL of anhydrous tetrahydrofuran and stirred for 2 minutes. 4- (4, 6-Dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (4.24g, 15.3mmol) and N-methylmorpholine (2.71g, 26.8mmol) were added and reacted at 25 ℃ for 8 hours. The residue is filtered off with suction and washed with a small amount of THF. Purification by column chromatography (petroleum ether: ethyl acetate ═ 5:1) gave 6.1g (88%) of the product as a white solid.
In the second step, lysine derivative 2 having a diyne group in the side chain (wherein l ═ 4) was synthesized:
it is characterized in that: obtaining the lysine derivative with side chain having diyne group through two-step reaction.
The preparation method comprises the following steps: product 1(2.31g, 3.5mmol) was dissolved in 15mL of dichloromethane, 1.40mL of triethylsilane was added, and 15mL of trifluoroacetic acid was added dropwise with stirring and the reaction was allowed to continue overnight. Evaporation of the solvent gave 1.42g of product (99%).
The third step: synthesizing a lysine derivative phenoxy monomer 3 with a diacetylene group on a side chain (wherein l ═ 4):
the preparation method comprises the following steps: product 2(1.75g, 3.5mmol) was added to 15mL of water and sodium carbonate (0.37g, 3.5mmol) was added and the mixture was heated to 45 ℃ and stirred for 2 min. S- (1, 3-benzothiazol-2-yl) -O-phenylthiocarbonate (1.2g, 4.2mmol) was dissolved in 45mL of tetrahydrofuran, added dropwise to the system, and reacted at 45 ℃ for 2 hours. The pH was adjusted to 3 with 20% sulfuric acid solution, extracted with EA and dried. Purification by column chromatography gave 1.0g of product (55%). The structure was characterized by nuclear magnetic hydrogen, carbon nuclear magnetic spectrum and high resolution Mass spectrometry (ESI-Mass), the results are shown in FIG. 1.
Example 2: diblock polypeptid MI-PEG45-b-PFOK11Preparation of
Mixing Fu-MI-PEG45-NH3 +Cl-Primary Macroamine initiator (45mg, 0.02mmol, 1 equiv.), FOK (199mg, 0.3mmol, 15 equiv.), and N, N-dimethylacetamide (1.5mL)And putting the mixture into a reaction tube filled with stirring magnetons. Stirring for 48h in an oil bath at the constant temperature of 70 ℃; benzyl isocyanate (20. mu.l) was added and reacted at 100 ℃ for 1 hour, and then the mixture was precipitated into an excess of methanol, and the precipitate was dissolved again with chloroform. The above dissolution-precipitation cycle was repeated 3 times. The final product was dried in a vacuum oven at room temperature overnight to give a white solid (96mg, yield: 57%). The white solid product was dispersed in 5ml of toluene and stirred at 100 ℃ for 6 hours. The mixture was then precipitated into an excess of methanol. The final product was dried in a vacuum oven at room temperature overnight to give a white solid (40mg, 87%). The hydrogen spectrum is shown in FIG. 3. The average DP of the PFOK blocks was determined to be 11 by hydrogen spectroscopy.
Example 3: preparation of microbubbles having a size of 2.4 microns
25mg of amphiphilic polypeptide MI-PEG45-PFOK11Example 2 was dissolved in 5ml of chloroform to prepare a clear solution. The solution was transferred to a rotary evaporator at 50 ℃ to remove the organic solvent and form a thin film. Then, 5ml of water was added and stirred at 50 ℃ for 48 hours, allowing the film to rehydrate to form a translucent milky white suspension forming micelles (fig. 4). The chamber was then evacuated and purged with perfluoropropane gas 3 times to ensure sonication in a perfluoropropane atmosphere with an amplitude of 40%. Finally, ultrasound time was 25 seconds and microbubbles of 2.4 microns could be formed. A confocal fluorescence microscopy brightfield photograph is shown in FIG. 5.
Example 4: PEG45-PFOK11Stability study of microvesicles under ultrasound stimulation
1mL of PEG45-PFOK11(example 2) the microbubble product was diluted by using 100mL of water and then infused into a disposable infusion set. When microbubbles enter the infusion tube, flow stops. Ultrasound images are obtained using a color doppler ultrasound system equipped with harmonic mode ultra-wideband nonlinear contrast imaging technology to obtain B-mode ultrasound and contrast enhanced ultrasound modes. The initial concentration of all microbubbles was fixed at 5 g/L. And calculating ultrasonic imaging signals of corresponding areas in the B ultrasonic mode and the contrast enhanced ultrasonic mode by using ImageJ (NIH). The results are shown in FIG. 6. It can be seen that our microbubbles were stimulated with ultrasoundThe stability is much higher than that of the conventional commercial microbubbles, so that the time for ultrasonic imaging is prolonged.
Example 5: MI-PEG45-PFOK11Microbubble in vivo ultrasound imaging studies
Female New Zealand white rabbits (provided by the animal center of university of medical, Anhui) were selected for animal experiments. Female New Zealand white rabbits (average body weight 2kg) were randomly selected and anesthetized by intraperitoneal injection of 10% chloral hydrate (3.5 mL/kg). Ultrasonic images are obtained by using a color Doppler ultrasonic system equipped with a harmonic mode ultra-wideband nonlinear contrast imaging technology to carry out B ultrasonic mode and contrast enhancement ultrasonic mode on the liver and the kidney of the rabbit. MI-PEG45-PFOK11The microbubble dispersion was injected through the ear vein at a dose of 0.5 mL/kg. Imagej (nih) was used to measure the ultrasound imaging signals of the corresponding region in the B-mode and the contrast enhanced ultrasound mode, respectively. As shown in fig. 7, the upper left image is rabbit liver imaging in the B-mode, the lower left image is rabbit kidney imaging in the B-mode, the upper right image is rabbit liver imaging in the contrast-enhanced ultrasound mode, and the lower right image is rabbit kidney imaging in the contrast-enhanced ultrasound mode, both having strong ultrasound signals. Is a microbubble which is highly stable and has a high imaging signal.
The present invention has been described in detail above, but the present invention is not limited to the specific embodiments described herein. It will be understood by those skilled in the art that other modifications and variations may be made without departing from the scope of the invention. The scope of the invention is defined by the appended claims.
Claims (10)
3. A method of preparing an amphiphilic polypeptide of formula (a) or (B), the method comprising:
(1) reacting a monomer of formula (a) or monomers of formulae (a) and (b) with a chain initiator of formula (c1) in a solvent, and
(2) adding benzyl isocyanate to react to obtain amphiphilic polypeptide shown in the formulas (A) and (B),
wherein each l is independently 2-4; i is 4 to 11; k is 4-400; n is 1 to 50; and m is 1 to 100.
4. A method of making an amphiphilic polypeptide of formula (C) or (D), the method comprising:
(1) reacting a monomer of formula (a) or monomers of formulae (a) and (b) with a chain initiator of formula (c2) in a solvent, and
(2) adding benzyl isocyanate to react to obtain amphiphilic polypeptide shown in the formulas (C) and (D),
wherein each l is independently 2-4; i is 4 to 11; k is 4-400; n is 1 to 50; and m is 1 to 100.
5. The method of claim 3 or 4, wherein
Preferably, the solvent in step (1) is selected from N, N-dimethylacetamide or dimethylsulfoxide;
preferably, the reaction temperature in step (1) is 60-90 ℃;
preferably, the reaction time in step (1) is 3 hours to 2 days;
preferably, the solvent in step (2) is selected from N, N-dimethylacetamide or chloroform;
preferably, the reaction temperature in step (2) is 50-110 ℃;
preferably, the reaction time in step (2) is 1 to 6 hours.
6. A polypeptidic microbubble prepared from the amphiphilic polypeptide of claim 1 or 2.
7. A method of making a polypeptidic microbubble, the method comprising: dissolving the amphiphilic polypeptide of claim 1 or 2 in an organic solvent, rotary evaporating the resulting organic solution to form a film, adding water for hydration, and finally subjecting the resulting system to ultrasound in an atmosphere containing a fluorine gas to obtain the polypeptidic microbubbles.
8. The method of claim 7, wherein
Preferably, the organic solvent is selected from chloroform, hexafluoroisopropanol or a mixture thereof in any proportion;
preferably, the concentration of the amphiphilic polypeptide in the organic solution is 0.1 mg/mL-100 mg/mL;
preferably, the volume of the water is 0.1 to 100 times of the volume of the organic solvent;
preferably, the temperature of hydration is 25-70 ℃, more preferably 50 ℃;
preferably, the time of hydration is 2 hours to 3 days, more preferably 2 days;
preferably, the fluorine-containing gas is selected from perfluoropropane, perfluorobutane and sulfur hexafluoride, more preferably perfluoropropane;
preferably, the sonication time is 10-60 seconds;
preferably, the ultrasound amplitude is 40% -70%.
9. The polypeptidic microvesicles of claim 6 or prepared according to the method of claim 7 or 8, wherein the microvesicles have a diameter ranging from 0.5 to 10 μ ι η.
10. Use of the polypeptidic microvesicles according to claim 6 or prepared according to the method of claim 7 or 8 for the preparation of a medicament for ultrasound imaging diagnosis.
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