CN115487321A - Contrast agent for cervical cancer ultrasonic contrast and preparation method thereof - Google Patents
Contrast agent for cervical cancer ultrasonic contrast and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/221—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by the targeting agent or modifying agent linked to the acoustically-active agent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—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
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- Health & Medical Sciences (AREA)
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Abstract
The invention discloses a contrast agent for cervical cancer ultrasonic contrast and a preparation method thereof, wherein the contrast agent comprises the following components in parts by weight: polyethylene glycol, dipalmitoyl phosphatidyl glycerol or salts thereof, distearoyl phosphatidyl choline and/or soybean phospholipid, the polypeptide NGKSAVLF and perfluoroolefin gas. The invention prepares the cervical cancer targeted ultrasonic contrast agent by designing a polypeptide sequence and combining with specific phospholipid, and improves the sensitivity and accuracy of ultrasonic diagnosis of cervical cancer.
Description
Technical Field
The invention belongs to the field of diagnostic reagents, and particularly relates to a contrast agent for cervical cancer ultrasonic contrast and a preparation method thereof.
Background
Cervical cancer is one of the common malignant tumors in gynecology, and in recent years, patients increase, and accurate diagnosis of cervical cancer has important significance on selection of treatment modes and treatment effects thereof. Ultrasound is a noninvasive and low-cost examination method, particularly the rapid development of an ultrasound contrast technique, can display the microcirculation change in a focus, and provides more reliable imaging information for clinic.
For example, zhang Jinhui, et al, examined the value of ultrasound imaging in cervical cancer stages (application of ultrasound imaging in cervical cancer stages [ J ]. Journal of clinical ultrasound medicine, 2013, 15 (6): 4.), selected 30 cervical cancer patients, pathologically confirmed and concurrently examined by conventional ultrasound and ultrasound imaging, observed the relationship between cervical lesions and surrounding tissues, analyzed their imaging data, compared the morphology of time-intensity curves and the difference of imaging parameters, and as a result, 28 ultrasound imaging diagnoses, with a diagnostic coincidence rate of 93.33%; the contrast features that when contrast medium is injected into 20.0 s, the lesion of cervical cancer is earlier than (16.0 s) and higher than the muscular layer development, and when contrast medium is injected into 26.7s, the contrast medium in the lesion is first eliminated and is low-enhanced and slightly higher-enhanced at the periphery, so that the infiltration range of cervical lesion can be clearly displayed. The research conclusion shows that the ultrasonic contrast of the cervical cancer has certain specificity and can assist clinical staging.
Ultrasonic contrast agents are a class of diagnostic agents that significantly enhance the ultrasonic detection signal, and generally comprise a microbubble structure with strong scattering properties for ultrasonic waves. In the above studies, the ultrasound contrast agent used was sononovacin. However, sononovine is a systemic contrast agent with no directional selection effect, and has low sensitivity and low specificity. In order to further enhance the targeted imaging enhancement effect of the ultrasonic contrast agent, the research on the targeted ultrasonic contrast agent is more and more, the existing targeted ultrasonic contrast agent usually utilizes antigen-antibody, biotin-avidin and other means to realize the targeted effect, and has the defects of complex preparation method, high requirement on storage and transportation conditions, high use cost and the like, so that the application of the targeted ultrasonic contrast agent is limited.
For example, chinese granted patent CN113041365B discloses a targeted ultrasound contrast agent for enhancing carotid arteriosclerosis ultrasound diagnosis and a preparation method thereof, the targeted ultrasound contrast agent for enhancing carotid arteriosclerosis ultrasound diagnosis is prepared from phospholipid containing hyaluronic acid and optionally mannose and derivatives thereof, and bio-inert gas, thereby realizing carotid arteriosclerosis targeting of the ultrasound contrast agent at low cost and enhancing the ultrasound imaging effect of carotid arteriosclerosis.
For another example, chinese granted patent CN111686263B discloses a targeted ultrasound contrast agent for enhancing the ultrasound diagnosis of arteriosclerosis of lower limbs and a preparation method thereof, the targeted ultrasound contrast agent for enhancing the ultrasound diagnosis of arteriosclerosis of lower limbs is prepared from an ultrasound contrast agent of an amino-modified lipid membrane and fibrin targeted polypeptide, so that the enrichment of the contrast agent at the affected part of arteriosclerosis of lower limbs is realized, the contrast duration is obviously prolonged, and the ultrasound imaging effect of arteriosclerosis of lower limbs is significantly enhanced.
However, how to develop a contrast agent capable of enhancing cervical cancer ultrasound contrast remains a technical problem to be solved by those skilled in the art.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a contrast agent capable of enhancing cervical cancer ultrasonic contrast. Specifically, in order to achieve the purpose of the present invention, the following technical solutions are proposed:
the invention relates to a contrast agent for cervical cancer ultrasonic contrast, which is characterized by comprising the following components in percentage by weight: polyethylene glycol, dipalmitoyl phosphatidyl glycerol or salts thereof, distearoyl phosphatidyl choline and/or soybean phospholipid, the polypeptide NGKSAVLF and perfluoroolefin gas.
In a preferred embodiment of the present invention, the perfluoroolefin gas is selected from the group consisting of: one or more of perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, perfluorooctane; more preferably, the perfluoroolefin gas is selected from the group consisting of: perfluoropropane and/or perfluorobutane.
In a preferred embodiment of the present invention, the weight ratio of the polyethylene glycol, dipalmitoyl phosphatidylglycerol or salts thereof, distearoyl phosphatidylcholine and/or soybean phospholipid, polypeptide NGKSAVLF is 2-4:3-6:2-4:0.2-0.5.
In a preferred embodiment of the present invention, the weight ratio of the polyethylene glycol, dipalmitoyl phosphatidylglycerol or salts thereof, distearoyl phosphatidylcholine and/or soybean phospholipid, polypeptide NGKSAVLF is 2-3:3-4:2-3:0.2-0.4.
In a preferred embodiment of the invention, the contrast agent is a freeze-dried powder injection in a sealed package.
In a preferred embodiment of the invention, the polyethylene glycol has an average weight average molecular weight of 1500 to 3000.
The invention also provides a preparation method of the ultrasonic contrast agent, which comprises the following steps:
(1) Adding polyethylene glycol, dipalmitoyl phosphatidyl glycerol or its salt, distearoyl phosphatidyl choline and/or soybean phospholipid into mixed solvent of ethanol water, homogenizing at 20-40 deg.C and 80-120MPa for 3-5 min, adding polypeptide NGKSAVLF, homogenizing for 0.5-2 min, and filtering with filter membrane to remove bacteria to obtain emulsion;
(2) Subpackaging the emulsion obtained in the step (1) by using a penicillin bottle, performing vacuum freeze drying to obtain freeze-dried powder, slowly filling perfluoroolefin gas in a vacuum state, slowly filling mixed gas of the perfluoroolefin gas and nitrogen, and sealing the penicillin bottle by using a rubber plug, wherein the volume ratio of the perfluoroolefin gas in the mixed gas is 40-60%;
(3) Packaging the sealed lyophilized powder into containers, and sterilizing by irradiation.
The invention has the beneficial effects
The cervical cancer targeting ultrasonic contrast agent is prepared by designing a polypeptide sequence and combining with specific phospholipid, so that the sensitivity and accuracy of ultrasonic diagnosis of cervical cancer are improved; on the other hand, the preparation method of the ultrasonic contrast agent prepared by the invention is simple and easy to implement, has low cost and is very favorable for wide introduction of clinical diagnosis; in another aspect, the ultrasonic contrast agent prepared by the invention is beneficial to reducing the clinical dosage of the ultrasonic contrast agent, and is beneficial to avoiding the side effect of the contrast agent and further reducing the use cost.
Drawings
Fig. 1 and 2 are graphs showing the results of ultrasonic diagnosis using the contrast agent prepared in example 1.
Detailed Description
In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless otherwise specified, the reagents used in the examples of the present invention are commercially available products, and all of them can be purchased from commercial sources. The polypeptide sequence NGKSAVLF is designed by the inventor and synthesized by Shanghai's workers by a solid-phase synthesis method.
Example 1: targeted ultrasonic contrast agent for ultrasonic diagnosis of cervical cancer
2 parts of polyethylene glycol 2000, 2 parts of distearoyl phosphatidylcholine (DSPC), 3 parts of dipalmitoyl phosphatidyl glycerol sodium, 0.2 part of polypeptide NGKSAVLF, 100 parts of 50 (v/v)% ethanol water mixed solvent and a proper amount of perfluoropropane, and the preparation method comprises the following steps:
(1) Adding polyethylene glycol 2000, distearoyl phosphatidylcholine and dipalmitoyl phosphatidyl glycerol sodium into a mixed solvent of ethanol water, homogenizing at 30 ℃ and 100MPa for 4 minutes, adding polypeptide, homogenizing for 1 minute, and filtering and sterilizing through a 220nm filter membrane to obtain emulsion;
(2) Subpackaging the emulsion obtained in the step (1) by using a penicillin bottle, performing vacuum freeze drying to obtain freeze-dried powder, slowly filling perfluoropropane to 55KPa in a vacuum state, slowly filling mixed gas of perfluoropropane and nitrogen to 1 standard atmospheric pressure, and sealing the penicillin bottle by using a rubber plug, wherein the volume of the perfluoropropane in the mixed gas accounts for 50%;
(3) Subpackaging the sealed lyophilized powder into containers for use 60 Co irradiation sterilization is carried out, and the targeted ultrasound contrast agent for cervical cancer ultrasound diagnosis is obtained.
Example 2:
the same as in example 1, except that soybean phospholipid was used in place of distearoyl choline.
Example 3:
the same as in example 1, except that the amount of the polypeptide NGKSAVLF used was adjusted to 0.1 part by weight.
Comparative example 1:
the same as in example 1, except that the polypeptide NGKSAVLF was not added.
Comparative example 2:
the same as in example 1 except that 5 parts by weight of distearoyl choline was used in place of the composition of distearoyl choline and dipalmitoyl sodium glycerophosphate.
Example 4: experiments in zoology
(1) And (3) cell culture: cultivation with DEME containing 10% calf serum at 37 ℃ and 5% CO 2 And conventionally culturing human cervical cancer Hela cells in a cell culture box.
(2) Constructing a transplantation tumor animal model: selecting 80 BALA/CA-nu nude mice, age of 5 weeks, weight of 15-18g, and injecting Hela cells of cervical cancer in logarithmic growth phase into the backs of the nude mice subcutaneously, each injection is 1 × 10 6 One cell per cell.
(3) Ultrasonic contrast examination: when the tumor body of the nude mice is observed by naked eyes to grow to about 0.5cm in diameter, the tumor-bearing nude mice are examined by ultrasonic radiography. 21 mice which were successfully molded were selected and randomly divided into a model group, a positive control group (Sonoweiwei), examples 1-3 and comparative examples 1-2, each group contained 0.1mL of ultrasound contrast medium (10 times the amount of physiological saline to form a suspension), and the model group was injected with the same amount of physiological saline. Using an ultrasonic diagnostic apparatus, measuring the frequency of a probe at 10MHz, and recording the ultrasonic contrast peak intensity ratio of tumor body parts of rats in each group, wherein:
peak intensity ratio = experimental group mean peak intensity/model group mean peak intensity.
(4) The experimental results are as follows: the experimental results are shown in table 1, and the experimental results show that the ultrasonic contrast agent of the invention has a more obvious ultrasonic enhancement effect, and the effect is higher than that of sononovacin in a positive control group. In particular, example 1 of the present invention showed more excellent targeting effect for cervical cancer.
TABLE 1 Effect of Targeted ultrasound contrast Agents on ultrasound diagnosis of cervical cancer
Example 5: clinical trial
1 cervical cancer patient collected in our hospital at 6 months in 2022 is diagnosed by pathological result diagnosis. The patient had no history of heart disease, contrast agents and other allergies, and signed an informed consent for a post-operative ultrasound contrast examination. The patient holds urine and takes the supine position, firstly, the patient carries out gynecological routine ultrasonic examination through the abdomen, records the position, the size, the shape, the echo characteristics and the like of the focus, and makes routine ultrasonic diagnosis. Selecting an optimal section capable of clearly displaying a focus, switching to a contrast mode, rapidly injecting 1.2ml of the contrast agent (prepared by 10 times of physiological saline into a suspension) prepared in the embodiment 1 into a bolus through an elbow vein, starting a timer, continuously observing 120S in real time, storing the process in an instrument hard disk, and playing back and analyzing a contrast image after the contrast is finished to make ultrasonic contrast diagnosis. Experimental results as shown in fig. 1-2, the experimental results show that the contrast of the contrast agent of the present invention can significantly improve the contrast at the cervix.
The above description is of the preferred embodiment of the present invention, but it is not intended to limit the present invention. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.
Claims (8)
1. A contrast agent for ultrasound contrast of cervical cancer, characterized in that the contrast agent comprises: polyethylene glycol, dipalmitoyl phosphatidyl glycerol or salts thereof, distearoyl phosphatidyl choline and/or soybean phospholipid, the polypeptide NGKSAVLF and perfluoroolefin gas.
2. The contrast agent according to claim 1, said perfluoroolefin gas being selected from the group consisting of: one or more of perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, perfluorooctane.
3. The contrast agent according to claim 1, the perfluoroolefin gas being selected from the group consisting of: perfluoropropane and/or perfluorobutane.
4. The contrast agent according to claim 1, wherein the weight ratio of polyethylene glycol, dipalmitoyl glycerol or a salt thereof, distearoyl choline and/or soybean lecithin, polypeptide NGKSAVLF is 2-4:3-6:2-4:0.2-0.5.
5. The contrast agent according to claim 1, wherein the weight ratio of the polyethylene glycol, dipalmitoyl phosphatidylglycerol or salt thereof, distearoyl phosphatidylcholine and/or soybean phospholipid, polypeptide NGKSAVLF is 2-3:3-4:2-3:0.2-0.4.
6. The contrast agent according to claim 1, which is a freeze-dried powder injection in sealed package.
7. The contrast agent according to claim 1, said polyethylene glycol having an average weight average molecular weight of 1500-3000.
8. A method for preparing an ultrasound contrast agent according to any of claims 1 to 7, comprising the steps of:
(1) Adding polyethylene glycol, dipalmitoyl phosphatidyl glycerol or its salt, distearoyl phosphatidyl choline and/or soybean phospholipid into mixed solvent of ethanol water, homogenizing at 20-40 deg.C and 80-120MPa for 3-5 min, adding polypeptide NGKSAVLF, homogenizing for 0.5-2 min, and filtering with filter membrane to remove bacteria to obtain emulsion;
(2) Subpackaging the emulsion obtained in the step (1) by using a penicillin bottle, performing vacuum freeze-drying to obtain freeze-dried powder, slowly filling perfluoroolefin gas in a vacuum state, slowly filling mixed gas of the perfluoroolefin gas and nitrogen, and sealing the penicillin bottle by using a rubber plug, wherein the perfluoroolefin gas accounts for 40-60% of the mixed gas by volume;
(3) Packaging the sealed lyophilized powder into containers, and sterilizing by irradiation.
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| CN202211153039.8A CN115487321B (en) | 2022-09-21 | 2022-09-21 | Contrast agent for ultrasound contrast of cervical cancer and preparation method thereof |
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| CN202211153039.8A CN115487321B (en) | 2022-09-21 | 2022-09-21 | Contrast agent for ultrasound contrast of cervical cancer and preparation method thereof |
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| CN115487321A true CN115487321A (en) | 2022-12-20 |
| CN115487321B CN115487321B (en) | 2023-10-31 |
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| CN105664188A (en) * | 2016-02-24 | 2016-06-15 | 袁哲 | Ultrasonic contrast agent of uterus oviduct tract and preparation method of ultrasonic contrast agent |
| CN107184994A (en) * | 2017-06-06 | 2017-09-22 | 山东省千佛山医院 | A kind of liver cancer Ultrasonic Diagnosis targeting agent and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105664188A (en) * | 2016-02-24 | 2016-06-15 | 袁哲 | Ultrasonic contrast agent of uterus oviduct tract and preparation method of ultrasonic contrast agent |
| CN107184994A (en) * | 2017-06-06 | 2017-09-22 | 山东省千佛山医院 | A kind of liver cancer Ultrasonic Diagnosis targeting agent and preparation method thereof |
Non-Patent Citations (1)
| Title |
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| 郑荣琴等: "超声造影新技术临床应用", vol. 1, 广东科技出版社, pages: 6 - 7 * |
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