WO2022001253A1 - 磁性超声造影剂组合物、磁性超声造影剂、磁性微泡超声造影剂及其制备方法 - Google Patents

磁性超声造影剂组合物、磁性超声造影剂、磁性微泡超声造影剂及其制备方法 Download PDF

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WO2022001253A1
WO2022001253A1 PCT/CN2021/084076 CN2021084076W WO2022001253A1 WO 2022001253 A1 WO2022001253 A1 WO 2022001253A1 CN 2021084076 W CN2021084076 W CN 2021084076W WO 2022001253 A1 WO2022001253 A1 WO 2022001253A1
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magnetic
contrast agent
surfactant
ultrasound contrast
microbubble
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PCT/CN2021/084076
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English (en)
French (fr)
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黄硕
郭雯雨
董飞宏
张珏
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南京超维景生物科技有限公司
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Priority to EP21833229.4A priority Critical patent/EP4046659A4/en
Publication of WO2022001253A1 publication Critical patent/WO2022001253A1/zh
Priority to US17/741,764 priority patent/US20220265869A1/en

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • 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
    • 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
    • A61K49/225—Microparticles, microcapsules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the field of biomedicine, in particular to a magnetic ultrasonic contrast agent composition, a magnetic ultrasonic contrast agent containing the magnetic ultrasonic contrast agent composition, a preparation method of a magnetic microbubble ultrasonic contrast agent, and the obtained magnetic microbubble ultrasonic contrast agent agent.
  • nano-controlled-release drug delivery systems such as tumor-responsive pH-controlled release systems, drug-loaded nanoparticles, etc.
  • these nano-drug delivery systems can realize the responsive release of drugs at tumor sites , but there are also some problems: such as insufficient local release, low tumor response sensitivity; especially in the high flow rate region, it is difficult to achieve drug enrichment delivery and visualized controllable release.
  • ultrasound-targeted microbubble destruction (UTMD) technology has been widely studied, because it has the ability to rupture microbubbles under the action of high-energy ultrasound to generate inertial cavitation, so that the loaded drug can be blasted in the target area at a fixed point and release the drug locally. And then play the therapeutic effect of targeted delivery of drugs.
  • UTMD technology in order to further realize drug enrichment delivery and visualized controllable release, there have been existing technologies on microbubble ultrasound contrast agents loaded with magnetic particles.
  • magnetic microbubbles currently used in UTMD technology The stability (cavitation threshold) of bubble ultrasound contrast agents is not ideal, and it is difficult to meet the requirements of targeted delivery in the circulation in vivo.
  • the purpose of the present invention is to overcome the problem of poor stability of the existing magnetic microbubble contrast agent, and to provide a magnetic ultrasonic contrast agent composition, a magnetic ultrasonic contrast agent containing the magnetic ultrasonic contrast agent composition, and a magnetic microbubble ultrasonic contrast agent A preparation method of the agent and a magnetic microbubble ultrasound contrast agent obtained therefrom.
  • the magnetic microbubble ultrasonic contrast agent obtained by the magnetic ultrasonic contrast agent composition of the present invention has high stability and can meet the requirements of targeted delivery in the circulation in vivo, thereby realizing local high-concentration targeted delivery of drugs.
  • the inventors of the present invention have found that the cavitation threshold of the magnetic microbubble ultrasonic contrast agent can be effectively increased by introducing specific components and proportions of surfactants into the magnetic ultrasonic contrast agent.
  • the inventors of the present invention also found that, in a particularly preferred embodiment, by specifically selecting poloxamer to cooperate with sodium citrate, the surface tension of the magnetic microbubble can be changed while filling the pores on the surface of the microbubble , thereby further improving the cavitation threshold and stability of the magnetic microbubble ultrasound contrast agent.
  • the first aspect of the present invention provides a magnetic ultrasound contrast agent composition
  • the magnetic ultrasound contrast agent composition comprises lipid, surfactant and magnetic nanoparticles, wherein, the surfactant is composed of surface active agent
  • the surface active agent A is composed of the surface active agent B, the surface active agent A is in a free state, and the surface active agent B is modified on the surface of the magnetic nanoparticles in a bound state;
  • the surface active agent A is a non-ferrous material with HLB value>8 Ionic surfactant
  • the surfactant B is a citric acid-based compound; relative to 1 mol of the lipid, the content of the surfactant A is 0.1-1 mol, and the content of the surfactant B is 0.1 -1 mol, the content of the magnetic nanoparticles is 0.05-0.5 mol.
  • the surfactant A is a nonionic surfactant with an HLB value>15; more preferably, a nonionic surfactant with an HLB value of 20-35.
  • the HLB value is determined by reversed-phase gas chromatography.
  • the hydrophilic-lipophilic balance value of non-ionic emulsifier is determined by reversed-phase gas chromatography, Synthetic Rubber Industry, 1991, 14 (6 ): 399-401.
  • the surfactant A is selected from one of poloxamers, poly(isobutylene-maleic anhydride) and poly(maleic anhydride-alt-1-octadecene) or More preferably, the surfactant A is a poloxamer.
  • the surface active agent B is citrate and/or citrate ester.
  • the surfactant A is poloxamer
  • the surfactant B is citrate
  • the ratio of the above-mentioned lipid, surfactant A agent, surfactant B agent and magnetic nanoparticles of the present invention can achieve better effects.
  • the The content of the surfactant A is 0.2-0.6 mol
  • the content of the surfactant B is 0.2-0.5 mol
  • the content of the magnetic nanoparticles is 0.1-0.3 mol; more preferably, relative to 1 mol of the Lipids, the content of the surfactant A is 0.3-0.5 mol, the content of the surfactant B is 0.3-0.4 mol, and the content of the magnetic nanoparticles is 0.18-0.25 mol.
  • the molar ratio of the magnetic nanoparticles to the surfactant B agent is 1:(0.6-2.5), more preferably 1:(1.2-1.8).
  • the 1:(1.2-1.8) is only a specific embodiment, and the upper limit can reach the saturation amount of the surfactant B agent on the magnetic nanoparticles.
  • the upper limit of 1:(0.6-2.5) in the molar ratio 1:2.5 is only for illustration. Under different circumstances, when the saturation amount is lower than 2.5 or higher than 2.5, the value can be taken within the range of 1: (0.6-saturation amount) according to the actual situation.
  • the magnetic microbubble ultrasound contrast agent of the present invention can be used as a drug delivery agent, based on the needs of production, transportation, etc., according to a specific embodiment, the ultrasound contrast agent composition of the present invention may not include drugs. .
  • the ultrasonic contrast agent composition further includes a drug, and the content of the drug is 100 parts by weight of the total weight of the lipid, surfactant and magnetic nanoparticles. 1-20 parts by weight, preferably 2-8 parts by weight.
  • the lipids may be conventional lipids used for ultrasound contrast agents in the art.
  • the lipid is a phospholipid.
  • the lipid is selected from one of polysorbate compounds and sorbitan monostearate or variety.
  • the lipid is preferably a combination of two or more substances.
  • the lipid is composed of sorbitan monostearate and polysorbate 80 in a molar ratio of 1:(0.5-2) (more preferably 1:(0.8-1.2)) than combined.
  • the magnetic nanoparticles can be selected from those commonly used in the field for magnetic ultrasound contrast agents.
  • the magnetic nanoparticles are superparamagnetic Fe 3 O 4 nanoparticles.
  • the particle size of the magnetic nanoparticles is 3-20 nm, preferably 7-10 nm.
  • the term "particle size” refers to the geometric spherical diameter of a single particle rather than an average value. When it is a range, it means that the particle size of the particles in the same material all fall within the range; The invention allows a certain error, that is, when the particle size of less than 5% of the total number is not within the required range, it is considered to meet the requirements.
  • the particle size of the magnetic nanoparticles is measured by transmission electron microscope, and the particle size of the microbubble is measured by optical microscope.
  • the medicament may be a medicament required for various practical treatments.
  • the drugs include, but are not limited to, paclitaxel, doxorubicin, bleomycin, and the like. All drugs that can be used in the ultrasound contrast agent in the art can be used in the present invention, and those skilled in the art can select them as needed.
  • the magnetic ultrasound contrast agent composition of the present invention may also contain other conventional adjuvants in the art, as long as the performance of other components is not adversely affected, those skilled in the art can choose, and the content of these other adjuvants can be Refer to the conventional content in the art.
  • the second aspect of the present invention provides a magnetic ultrasound contrast agent, which contains the magnetic ultrasound contrast agent composition described in the first aspect of the present invention.
  • the ultrasound contrast agent contains a large number of gas microbubbles.
  • the particle size of the gas microbubbles is 0.3-5 ⁇ m, more preferably 0.8-3 ⁇ m.
  • the gas in the gas microbubbles is an inert gas, which can be conventional gas used for microbubble ultrasound contrast agents in the art, for example, a gas selected from perfluoropropane, perfluorobutane and sulfur hexafluoride one or more.
  • the outer shell of the gas microbubble contains the ultrasound contrast agent composition according to the first aspect of the present invention.
  • the ultrasound contrast agent does not contain gas microbubbles.
  • the ultrasonic contrast agent in this state is called an ultrasonic contrast agent film-forming agent in the art.
  • the ultrasonic contrast agent film-forming agent can be obtained by applying a certain mechanical force, such as ultrasonic cavitation, which can be used clinically and contains a large amount of gas. Microbubble ultrasound contrast agent.
  • ultrasound contrast agent also includes ultrasound contrast agent film-forming agents.
  • the ultrasound contrast agent is composed of a continuous phase and a dispersed phase
  • the continuous phase may be a conventional continuous phase used in the art for preparing ultrasound contrast agents, such as a phosphate (PBS) buffer solution
  • PBS phosphate
  • the dispersed phase contains the ultrasonic contrast agent composition described in the first aspect of the present invention, which can be gas microbubbles (ie, to form an ultrasound contrast agent) or lipid droplets (ie, to form an ultrasound contrast agent film-forming solution).
  • the third aspect of the present invention provides a method for preparing a magnetic microbubble ultrasound contrast agent, the raw materials used include the magnetic ultrasound contrast agent composition described in the first aspect of the present invention, and the method includes the following steps:
  • step (2) mixing the medicine with the material obtained in step (1), feeding gas into the material obtained by mixing, and performing ultrasonic cavitation to form the first microbubble suspension;
  • the specific selection and ratio of the raw materials used are in accordance with the definitions in the magnetic ultrasound contrast agent composition described in the first aspect of the present invention, which will not be repeated here.
  • the first contact makes the mixing of the lipid and surfactant A more sufficient, so that the core-shell structure of the microvesicles of the first microvesicle suspension obtained in step (2) is more complete Stable; preferably, the conditions of the first contact include: a temperature of 110-130° C. and a time of 8-16 minutes.
  • step (2) preferably, the mixing is performed at a temperature of 30-50°C.
  • the material obtained in step (1) can be cooled to this temperature range before mixing with the drug.
  • the conditions of the ultrasonic cavitation include: the ultrasonic power is 8-12kW, and the time is 1-8min; more preferably, the conditions of the ultrasonic cavitation include: the ultrasonic power is 9- 11kW, time is 2-4min.
  • step (3) preferably, before carrying out the loading and positive charge treatment, the first microbubble suspension is left standing and centrifuged to remove the microbubbles with larger particle size in the upper layer,
  • the particle size of the microvesicles in the first microvesicle suspension after centrifugation is 0.3-5 ⁇ m, more preferably 0.8-3 ⁇ m.
  • the process of loading positive charge treatment includes: contacting the first microbubble suspension with an aqueous solution of a cationic reagent.
  • the volume ratio of the cationic reagent aqueous solution to the first microbubble suspension is (0.8-1.2):1.
  • the concentration of cations in the cationic reagent aqueous solution is 0.5-2 mg/L.
  • the method of contacting the first microbubble suspension with the cationic reagent aqueous solution is not particularly limited, and preferably, ultrasonic oscillation is used, the ultrasonic power is 170-420W, and the time is 20-40min.
  • the positive charges carried by the cationic agent are adsorbed on the surface of the microbubbles in the first microbubble suspension.
  • step (3) preferably, the method further comprises: leaving the material in contact with the cationic reagent to stand for stratification, taking the upper layer material and washing it with a buffer solution to obtain a second microbubble suspension with a positive surface charge liquid.
  • the upper layer material obtained after the static layering is an oily solution containing the first microbubble suspension
  • the lower layer material is an aqueous solution.
  • the buffer solution may be a conventional acid-base buffer solution in the art, for example, a phosphate (PBS) buffer solution.
  • the conditions of the second contact include: ultrasonically oscillating the material, the ultrasonic power is 170-420W, and the time is 15-50min; more preferably, the ultrasonic power is 250-350W , the time is 20-40min.
  • the magnetic nanoparticles are superparamagnetic nanoparticles.
  • the surface-modified magnetic nanoparticles with surface active agent B can be prepared in a conventional manner in the art.
  • the preparation method of the magnetic nanoparticles whose surface is modified with the surfactant B agent comprises the following steps: ultrasonically oscillating the mixture of the magnetic nanoparticles and the aqueous solution of the surfactant B agent, and the ultrasonic power is 170-420W, The time is 8-30 min, and then the solid magnetic nanoparticles are separated; repeated 2-5 times; the magnetic nanoparticles whose surfaces are modified with the surface active agent B are obtained.
  • the amount of the magnetic nanoparticles and the surfactant B is such that the weight ratio satisfies the limitation in the magnetic ultrasound contrast agent composition described in the first aspect of the present invention.
  • the magnetic nanoparticles are superparamagnetic nanoparticles, which can be obtained commercially or prepared.
  • the preparation method preferably includes: thermally decomposing the mixture of iron precursor and solvent under oxygen-free and anhydrous conditions to obtain superparamagnetic nanoparticles; then subjecting the superparamagnetic nanoparticles to magnetic Suction purification.
  • the preparation method preferably comprises: carrying out a thermal decomposition reaction of the mixture obtained by ferric acetylacetonate and triethylene glycol in a molar ratio of (2.5-3.5): 1 under anoxic and anhydrous conditions, and the thermal decomposition reaction
  • the temperature is 270-290° C. and the time is 0.8-1.2 h to obtain superparamagnetic nanoparticles; and then the superparamagnetic nanoparticles are purified by magnetic absorption.
  • the present invention has at least the following advantages compared with the prior art: the microbubble ultrasound contrast agent obtained from the ultrasound contrast agent composition of the present invention can be controlled by an external magnetic field to realize the enrichment and response of the drug at a specific site At the same time, sufficient stability can be ensured, and stability can be maintained in the state of local high flow rate, and the performance of the ultrasound contrast agent of the present invention is controllable; therefore, the present invention enables stable and reliable targeted drug delivery in the state of local high flow rate. possible.
  • Fig. 1 is the optical microscope electron microscope picture of the ultrasound contrast agent S1 obtained in Example 1;
  • Figure 2(a) is an ultrasound image of the enriched grayscale of the microbubble ultrasound contrast agent D4 obtained in Comparative Example 4;
  • FIG. 2( b ) is an ultrasound image of the enriched grayscale of the microbubble ultrasound contrast agent S1 obtained in Example 1.
  • FIG. 2( b ) is an ultrasound image of the enriched grayscale of the microbubble ultrasound contrast agent S1 obtained in Example 1.
  • Triethylene glycol and iron acetylacetonate are mixed in a molar ratio of 3:1. Heating at a high temperature of 280 °C for 1 h, superparamagnetic Fe 3 O 4 nanoparticles were obtained, and the particle size was 7-10 nm by transmission electron microscopy;
  • step (c) mixing the superparamagnetic nanoparticles obtained in step (b) with the surfactant B agent aqueous solution (sodium citrate solution) at a molar ratio of 1:1.5, and oscillating for 15 min under ultrasonic conditions of 300 kW, repeating 3 times;
  • surfactant B agent aqueous solution sodium citrate solution
  • the difference is that the amount of sodium citrate is changed so that the molar ratio of superparamagnetic nanoparticles to surfactant B is 1:0.5.
  • Sorbitan monostearate lipid
  • polysorbate 80 lipid
  • step (2) mixing the material obtained in step (1) with paclitaxel (chemotherapeutic drug) in a weight ratio of 100:5; using ultrasonic cavitation method, at room temperature, perfluorocarbon gas is introduced into the material obtained by mixing, Ultrasonic cavitation was carried out for 3min under the power of 10kW, cooled to room temperature to form the first microbubble suspension;
  • paclitaxel chemotherapeutic drug
  • the obtained magnetic microbubble ultrasound contrast agent S1 was observed under an optical microscope, and the obtained results are shown in FIG. 1 . It can be seen from Figure 1 that the contrast agent is densely covered with microbubbles with a particle size of about 1 ⁇ m. The microbubbles have a narrow particle size distribution and no obvious impurities in the solution, which can meet the contrast requirements of ultrasonic contrast agents.
  • Sorbitan monostearate lipid
  • polysorbate 80 lipid
  • step (2) mixing the material obtained in step (1) with paclitaxel (chemotherapeutic drug) in a weight ratio of 100:8; using ultrasonic cavitation method, at room temperature, perfluorocarbon gas is introduced into the material obtained by mixing, Ultrasonic cavitation was carried out for 3min under the power of 10kW, cooled to room temperature to form the first microbubble suspension;
  • paclitaxel chemotherapeutic drug
  • Sorbitan monostearate lipid
  • polysorbate 80 lipid
  • step (2) mixing the material obtained in step (1) with paclitaxel (chemotherapeutic drug) in a weight ratio of 100:2; using ultrasonic cavitation method, at room temperature, perfluorocarbon gas is introduced into the material obtained by mixing, Ultrasonic cavitation was carried out for 3 min under the power of 10 kW, and cooled to room temperature to form the first microbubble suspension;
  • paclitaxel chemotherapeutic drug
  • the difference is that the ratio of two lipids is changed, specifically, the molar ratio of sorbitan monostearate and polysorbate 80 added is 1:2.5.
  • Example 1 The procedure of Example 1 was followed except that the poloxamer was replaced by the same molar amount of poly(isobutylene-maleic anhydride) (Sigma-Aldrich, 531278-250G).
  • Example 1 The procedure of Example 1 was followed, except that the poloxamer was replaced by the same molar amount of poly(maleic anhydride-alt-1-octadecene) (Sigma-Aldrich, 419117-250G).
  • Example 1 The procedure of Example 1 was followed, except that no poloxamer was added.
  • Example 2 Carry out with reference to the method of Example 1, the difference is that the surfactant A agent poloxamer used in Example 1 is replaced with glycerol monostearate (Tianwei Taida, RH30299-100g).
  • the difference is that the sodium citrate-modified superparamagnetic nanoparticles obtained in Preparation Example 1 are replaced with the same amount of superparamagnetic nanoparticles modified with poloxamer on the surface obtained in Comparative Preparation Example 1. Fe 3 O 4 magnetic nanoparticles.
  • the difference is, do not add the superparamagnetic nanoparticles modified by sodium citrate, namely do not carry out steps (3) and (4), the surface of the obtained step (2) is positively charged first.
  • a microbubble suspension is the final microbubble ultrasound contrast agent, denoted as D4.
  • the stability of the ultrasound contrast agent in vivo is reflected by the half-life of the ultrasound contrast agent, and the longer the half-life, the higher the stability.
  • the specific test methods include (with rabbits as the object):
  • Carry out in vitro enrichment experiments, and the specific test methods include:
  • the same concentration (10 6 /mL) of the ultrasound contrast agent of the example and the comparative example was injected respectively, and at a static flow rate (0mL/h) in vitro enrichment and explosion experiments of magnetic microbubbles were carried out.
  • the ultrasonic imaging probe adopts a linear array probe with 196 array elements, a center frequency of 12MHz and a frequency bandwidth of 6MHz.
  • the mechanical index of the ultrasonic wave emitted by the ultrasonic probe is controlled (the control range is MI ⁇ 0.1), and the magnetic properties of the magnetic microbubbles under different mechanical indices are observed. .
  • the ultrasonic image shows that the grayscale signals of the magnetic microbubbles at the magnet end are uniformly distributed in the cellulose tube), it is considered non-magnetic.
  • the ultrasound contrast agent composition and ultrasound contrast agent of the present invention can take into account longer half-life, higher blasting mechanical index and good magnetic properties, so that the magnetic microbubble ultrasound contrast agent can meet the requirements of in vivo circulation Requirements for targeted delivery.

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Abstract

一种磁性超声造影剂组合物、磁性超声造影剂、磁性微泡超声造影剂及其制备方法。该磁性超声造影剂组合物包括脂质、表面活性剂和磁性纳米颗粒，其中，表面活性剂由表面活性A剂和表面活性B剂组成，表面活性A剂呈游离态，表面活性B剂呈结合态修饰于磁性纳米颗粒的表面；表面活性A剂HLB值>8；相对于1mol的所述脂质，表面活性A剂的含量为0.1-1mol，表面活性B剂的含量为0.1-1mol，磁性纳米颗粒的含量为0.05-0.5mol。磁性超声造影剂组合物所得到的磁性微泡超声造影剂具有较高的稳定性，能够满足体内循环靶向递送的要求，从而实现药物的局部高浓度靶向递送。

Description

磁性超声造影剂组合物、磁性超声造影剂、磁性微泡超声造影剂及其制备方法 技术领域
本发明涉及生物医药领域，具体涉及一种磁性超声造影剂组合物、含有该磁性超声造影剂组合物的磁性超声造影剂、磁性微泡超声造影剂的制备方法及其得到的磁性微泡超声造影剂。
背景技术
目前，治疗各种癌症的特效药物通常难溶于水、副作用大、易产生耐药。有研究表明，靶向递送可以解决上述问题。近年来，各国科学家在纳米控释递药体系领域进行了多方面的研究，如肿瘤响应型pH控释系统、载药纳米颗粒等，虽然这些纳米递药系统可实现药物在肿瘤部位的响应释放，但亦存在一些问题：如局部释放量不足、肿瘤响应灵敏度低；尤其是在高流速区域不易实现药物富集递送以及可视化的可控释放等。
近年来，超声靶向微泡破坏(UTMD)技术受到了广泛研究，因为其具有能够在高能量超声波作用下微泡破裂发生惯性空化，从而可以负载药物在靶区定点爆破并局部释放药物，进而发挥靶向递送药物的治疗作用。在UTMD技术的基础上，为了进一步实现药物富集递送以及可视化的可控释放，已经有现有技术对负载有磁性粒子的微泡超声造影剂进行了研究，然而目前用于UTMD技术的磁性微泡超声造影剂的稳定性(空化阈值)并不理想，难以满足体内循环靶向递送的要求。
因此，研究一种稳定性较为理想的磁性微泡超声造影剂对于疾病的治疗特别是癌症的治疗将具有非常重要的意义。
发明内容
本发明的目的在于克服现有的磁性微泡造影剂稳定性较差的问题，提供一种磁性超声造影剂组合物、含有该磁性超声造影剂组合物的磁性超声造影剂、磁性微泡超声造影剂的制备方法及其得到的磁性微泡超声造影剂。
本发明的磁性超声造影剂组合物所得到的磁性微泡超声造影剂具有较高的稳定性，能够满足体内循环靶向递送的要求，从而实现药物的局部高浓度靶向递送。
本发明的发明人发现，通过向磁性超声造影剂中引入特定成分和配比的表面活性剂，能够有效地提高磁性微泡超声造影剂的空化阈值。本发明的发明人还发现，在一种特别优选的实施方式中，通过特定地选用泊洛沙姆和柠檬酸钠相配合，能够在改变磁性微泡的表面张力的同时填补微泡表面的孔洞，从而进一步提高磁性微泡超声造影剂的空化阈值和稳定性。
为了实现上述目的，本发明第一方面提供了一种磁性超声造影剂组合物，该磁性超声造影剂组合物包括脂质、表面活性剂和磁性纳米颗粒，其中，所述表面活性剂由表面活性A剂和表面活性B剂组成，所述表面活性A剂呈游离态，所述表面活性B剂呈结合态修饰于所述磁性纳米颗粒的表面；所述表面活性A剂为HLB值>8的非离子型表面活性剂，所述表面活性B剂为柠檬酸基化合物；相对于1mol的所述脂质，所述表面活性A剂的含量为0.1-1mol，所述表面活性B剂的含量为0.1-1mol，所述磁性纳米颗粒的含量为0.05-0.5mol。
优选地，所述表面活性A剂为HLB值>15的非离子型表面活性剂；更优选为HLB值为20-35的非离子型表面活性剂。
在本发明中，所述HLB值通过反相气相色谱法测定，参照江朝学等，用反相气相色谱法测定非离子型乳化剂的亲水亲油平衡值，合成橡胶工业，1991,14(6):399-401。
根据一种具体的实施方式，所述表面活性A剂选自泊洛沙姆、聚(异丁 烯-马来酸酐)和聚(马来酸酐-alt-1-十八碳烯)中的一种或多种；更优选地，所述表面活性A剂为泊洛沙姆。
优选地，所述表面活性B剂为柠檬酸盐和/或柠檬酸酯。
根据本发明一种具体实施方式，所述表面活性A剂为泊洛沙姆，且所述表面活性B剂为柠檬酸盐。
本发明的上述脂质、表面活性A剂、表面活性B剂和磁性纳米颗粒的配比即能够实现较好的效果，为了进一步提高稳定性，优选地，相对于1mol的所述脂质，所述表面活性A剂的含量为0.2-0.6mol，所述表面活性B剂的含量为0.2-0.5mol，所述磁性纳米颗粒的含量为0.1-0.3mol；更优选地，相对于1mol的所述脂质，所述表面活性A剂的含量为0.3-0.5mol，所述表面活性B剂的含量为0.3-0.4mol，所述磁性纳米颗粒的含量为0.18-0.25mol。
优选地，所述磁性纳米颗粒与所述表面活性B剂的摩尔比为1：(0.6-2.5)，更优选为1：(1.2-1.8)。所述1：(1.2-1.8)仅为一种具体实施方式，上限可以达到所述表面活性B剂在磁性纳米颗粒上的饱和量，同样地，摩尔比中1：(0.6-2.5)的上限1:2.5也仅为示意，在不同情况下，当饱和量低于2.5时或高于2.5时，均可以根据实际情况在1：(0.6-饱和量)范围内取值。
可以理解的是，虽然本发明的磁性微泡超声造影剂能够作为药物递送剂，但是基于生产、运输等的需要，根据一种具体实施方式，本发明的超声造影剂组合物中可以不包括药物。
根据本发明另一种具体实施方式，所述超声造影剂组合物还包括药物，相对于100重量份的所述脂质、表面活性剂和磁性纳米颗粒的重量之和，所述药物的含量为1-20重量份，优选为2-8重量份。
在本发明中，所述脂质可以为本领域常规的用于超声造影剂的脂类。优选地，所述脂质为磷脂。为了与本发明的超声造影剂组合物中的其他成分发挥更好的协同作用，优选地，所述脂质选自聚山梨醇酯类化合物和山梨醇酐单硬脂酸酯中的一种或多种。
在本发明中，所述脂质优选为两种及多种物质的组合。根据一种优选的具体实施方式，所述脂质由山梨醇酐单硬脂酸酯和聚山梨醇酯80以1：(0.5-2)(更优选为1：(0.8-1.2))的摩尔比组合而成。
在本发明中，所述磁性纳米颗粒可以选用本领域用于磁性超声造影剂常用的磁性纳米颗粒。优选地，所述磁性纳米颗粒为超顺磁性Fe 3O 4纳米颗粒。
在本发明中，优选地，所述磁性纳米颗粒的粒径为3-20nm，优选为7-10nm。
在本发明中，术语“粒径”指的是单个颗粒的几何学球形直径而并非平均值，当为范围时，指同一物料中的该种颗粒的粒径均落在该范围内；同时本发明允许一定的误差，即当占总数量不到5％的颗粒粒径不在要求的范围内时也视为满足要求。本发明中磁性纳米颗粒的粒径通过透射电镜测量，微泡的粒径通过光学显微镜测量。
在本发明中，所述药物可以为各种实际治疗所需要的药物。例如，所述药物包括但不限于紫杉醇、阿霉素、博来霉素等。本领域能够用于超声造影剂中的药物均可以用于本发明中，本领域技术人员能够根据需要进行选择。
本发明的磁性超声造影剂组合物中还可以含有其他本领域的常规助剂，只要不会对其他组分的性能产生不利影响，本领域技术人员均可以进行选择，这些其他助剂的含量可以参照本领域的常规含量。
本发明第二方面提供了一种磁性超声造影剂，该磁性超声造影剂中含有本发明第一方面所述的磁性超声造影剂组合物。
根据本发明一种具体实施方式，所述超声造影剂中含有大量的气体微泡。
在这种具体实施方式中，优选地，所述气体微泡的粒径为0.3-5μm，更优选为0.8-3μm。
优选地，所述气体微泡中的气体为惰性气体，可以为本领域中常规的用于微泡超声波造影剂的气体，例如选自全氟丙烷、全氟丁烷和六氟化硫中的 一种或多种。所述气体微泡的外壳即含有本发明第一方面所述的超声造影剂组合物。
根据本发明另一种具体实施方式，所述超声造影剂中不含有气体微泡。通常本领域将这种状态下的超声造影剂称为超声造影剂成膜剂，该超声造影剂成膜剂通过被施加一定的机械力，例如超声空化，可以得到能够临床使用的含有大量气体微泡的超声造影剂。
在本发明中，术语“超声造影剂”也包括超声造影剂成膜剂。
根据本发明的具体实施方式，所述超声造影剂由连续相和分散相组成，所述连续相可以为本领域常规的用来制备超声造影剂的连续相，例如磷酸盐(PBS)缓冲溶液；所述分散相中含有本发明第一方面所述的超声造影剂组合物，可以为气体微泡(即形成超声造影剂)或者脂质液滴(即形成超声造影剂成膜溶液)。
本发明第三方面提供了一种制备磁性微泡超声造影剂的方法，所用原料包括本发明第一方面所述的磁性超声造影剂组合物，所述方法包括以下步骤：
(1)将脂质和表面活性A剂进行第一接触；
(2)将药物与步骤(1)所得物料混合，向所述混合所得物料中通入气体，进行超声空化形成第一微泡混悬液；
(3)将所述第一微泡混悬液进行负载正电荷化处理，得到表面带正电荷的第二微泡混悬液；
(4)将表面修饰有表面活性B剂的磁性纳米颗粒与所述第二微泡混悬液进行第二接触。
在本发明第三方面的方法中，所用到的原料的具体选择和配比均按照本发明第一方面所述的磁性超声造影剂组合物中的限定进行，在此不再赘述。
在步骤(1)中，所述第一接触使得所述脂质和表面活性A剂的混合更加充分，从而使得步骤(2)所得的第一微泡混悬液的微泡的核壳结构更加 稳定；优选地，所述第一接触的条件包括：温度为110-130℃，时间为8-16min。
在步骤(2)中，优选地，所述混合在30-50℃的温度下进行。例如，可以将步骤(1)所得物料先降温至该温度范围在与所述药物进行所述混合。
在步骤(2)中，优选地，所述超声空化的条件包括：超声功率为8-12kW，时间为1-8min；更优选地，所述超声空化的条件包括：超声功率为9-11kW，时间为2-4min。
在步骤(3)中，优选地，在进行所述负载正电荷化处理之前，先将所述第一微泡混悬液进行静置和离心分离，以除去上层粒径较大的微泡，优选使得离心分离后的第一微泡混悬液中微泡的粒径为0.3-5μm，更优选为0.8-3μm。
在步骤(3)中，优选地，所述负载正电荷化处理的过程包括：将所述第一微泡混悬液与阳离子试剂水溶液进行接触。
优选地，所述阳离子试剂水溶液与所述第一微泡混悬液的体积比为(0.8-1.2)：1。优选地，所述阳离子试剂水溶液中的阳离子的浓度为0.5-2mg/L。
所述将第一微泡混悬液与阳离子试剂水溶液进行接触的方式没有特别的限定，优选地，采用超声振荡的方式进行，超声功率为170-420W，时间为20-40min。通过该接触，使得阳离子试剂所携带的正电荷吸附于所述第一微泡混悬液中微泡的表面。
在步骤(3)中，优选地，所述方法还包括：将与阳离子试剂接触后的物料静置分层，取上层物料并用缓冲溶液进行洗涤，得到表面带正电荷的第二微泡混悬液。
所述静置分层后所得上层物料为含有所述第一微泡混悬液的油性溶液，下层物料为水性溶液。取上层溶液之后用缓冲溶液进行洗涤2-5次以除去过量的阳离子试剂。所述缓冲溶液可以为本领域常规的酸碱缓冲溶液，例如可以为磷酸盐(PBS)缓冲溶液。
在步骤(4)中，优选地，所述第二接触的条件包括：对物料进行超声 振荡，超声功率为170-420W，时间为15-50min；更优选地，所述超声功率为250-350W，所述时间为20-40min。
在本发明中，优选地，所述磁性纳米颗粒为超顺磁性纳米颗粒。
在本发明中，所述表面修饰有表面活性B剂的磁性纳米颗粒可以按照本领域常规的方式进行制备获得。优选地，所述表面修饰有表面活性B剂的磁性纳米颗粒的制备方法包括以下步骤：将磁性纳米颗粒与所述表面活性B剂的水溶液的混合物料进行超声振荡，超声功率为170-420W，时间为8-30min，然后分离出固体磁性纳米颗粒；重复2-5次；得到表面修饰有表面活性B剂的磁性纳米颗粒。
所述磁性纳米颗粒与所述表面活性B剂的用量使得重量比满足本发明第一方面所述的磁性超声造影剂组合物中的限定。
在本发明中，优选地，所述磁性纳米颗粒为超顺磁性纳米颗粒，可以商购获得，也可以制备得到。当通过制备得到时，制备方法优选包括：将铁前驱物与溶剂的混合物料在无氧无水条件下进行热分解反应，得到超顺磁性纳米颗粒；然后将所述超顺磁性纳米颗粒进行磁吸提纯。
更优选地，所述制备方法优选包括：将乙酰丙酮铁与三甘醇以(2.5-3.5)：1的摩尔比得到的混合物料在无氧无水条件下进行热分解反应，该热分解反应的温度为270-290℃，时间为0.8-1.2h，得到超顺磁性纳米颗粒；然后将所述超顺磁性纳米颗粒进行磁吸提纯。
通过上述技术方案，本发明与现有技术相比至少具有以下优势：本发明的超声造影剂组合物得到的微泡超声造影剂能够通过外加磁场的控制而实现药物在特定部位的富集和响应释放，同时能够保证足够的稳定性，能够在局部高流速状态下保持稳定，并且本发明的超声造影剂的性能可控；因此本发明使得在局部高流速状态下稳定可靠的靶向药物递送成为可能。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各 个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
附图说明
图1是实施例1所得超声造影剂S1的光学显微镜电镜图；
图2(a)是对比例4所得的微泡超声造影剂D4的富集灰度的超声图像；
图2(b)是实施例1所得的微泡超声造影剂S1的富集灰度的超声图像。
具体实施方式
以下将通过实施例对本发明进行详细描述。本发明所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
在没有特别说明的情况下，以下实施例所用的物料均为商购的分析纯。
以下制备例用于制备实施例中所使用的表面修饰有表面活性B剂的磁性纳米颗粒，以下制备例仅为本发明的具体实施方式，不作为本发明的限定。
制备例1
(a)将三甘醇、乙酰丙酮铁按照摩尔比例3：1混合。在高温280℃加热1h，得到超顺磁性Fe 3O 4纳米颗粒，并利用透射电镜观察粒径为7-10nm；
(b)将所述超顺磁性纳米颗粒进行磁吸提纯；
(c)将步骤(b)所得超顺磁性纳米颗粒与表面活性B剂水溶液(柠檬酸钠溶液)以摩尔比1:1.5混合，在300kW的超声条件下振荡15min，重复3次；
得到表面修饰有柠檬酸钠的超顺磁性Fe 3O 4纳米颗粒。
制备例2
参照制备例1的方法进行，所不同的是，改变柠檬酸钠的用量，使得超顺磁性纳米颗粒与表面活性B剂的摩尔比为1：1.8。
制备例3
参照制备例1的方法进行，所不同的是，改变柠檬酸钠的用量，使得超顺磁性纳米颗粒与表面活性B剂的摩尔比为1：1.2。
制备例4
参照制备例1的方法进行，所不同的是，改变柠檬酸钠的用量，使得超顺磁性纳米颗粒与表面活性B剂的摩尔比为1：0.5。
对比制备例1
参照制备例1的方法进行，所不同的是，将柠檬酸钠替换为相同摩尔量的泊洛沙姆，得到表面修饰有泊洛沙姆的超顺磁性Fe 3O 4纳米颗粒。
实施例1
按照以下步骤进行：
(1)将山梨醇酐单硬脂酸酯(脂质)、聚山梨醇酯80(脂质)以及泊洛沙姆F68(德国BASF，P21489-25G，表面活性A剂，HLB值＝29)按照1：1：1.5的摩尔比混合均匀，取10mL置于120℃灭菌锅中高温高压12min，之后连续搅拌冷却至40度；
(2)将步骤(1)所得物料与紫杉醇(化疗药物)以100:5的重量比混合；采用超声空化法，在室温下向所述混合所得物料中通入全氟化碳气体，在10kW的功率下超声空化3min，冷却至室温，形成第一微泡混悬液；
(3)将所述第一微泡混悬液静置12h后离心除去上层粒径较大的微泡，通过光镜测量微泡的粒径分布在0.8-3μm的范围内；然后向其中缓慢加入10 mL聚乙烯亚胺溶液(浓度为1mg/L)，在300kW下超声振荡，吸附30min后，置于4℃冰箱中静置分层，弃下层清液；然后将上层液体用PBS缓冲液反复洗涤3次，除去过量的聚电解质，得到表面带正电荷的第二微泡混悬液；
(4)向所述第二微泡混悬液中缓慢加入10mL制备例1所得的柠檬酸钠修饰的超顺磁性纳米颗粒的水溶液(其中超顺磁性纳米颗粒的量与第二微泡混悬液中脂质的摩尔比为0.25:1)，在300kW下超声振荡，吸附30min后，置于4℃冰箱中静置分层，弃下层清液；然后将上层液体用PBS缓冲液反复洗涤3次，得到磁性微泡超声造影剂，记为S1。
将所得磁性微泡超声造影剂S1进行光学显微镜观测，所得结果如图1所示。从图1中可以看出该造影剂密布有粒径为1μm左右的微泡，微泡的粒径分布窄且溶液中没有明显的杂质，能够满足超声造影剂的造影需求。
实施例2
按照以下步骤进行：
(1)将山梨醇酐单硬脂酸酯(脂质)、聚山梨醇酯80(脂质)以及泊洛沙姆F127(西格玛奥德里奇，P2443-250G，表面活性A剂，HLB值＝23)按照1：0.8：1.35的摩尔比混合均匀，取10mL置于110℃灭菌锅中高温高压16min，之后连续搅拌冷却至40度；
(2)将步骤(1)所得物料与紫杉醇(化疗药物)以100:8的重量比混合；采用超声空化法，在室温下向所述混合所得物料中通入全氟化碳气体，在10kW的功率下超声空化3min，冷却至室温，形成第一微泡混悬液；
(3)将所述第一微泡混悬液静置12h后离心除去上层粒径较大的微泡，通过光镜测量微泡的粒径分布在0.8-3μm的范围内；然后向其中缓慢加入10mL聚乙烯亚胺溶液(浓度为1.5mg/L)，在250kW下超声振荡，吸附40min后，置于4℃冰箱中静置分层，弃下层清液；然后将上层液体用PBS缓冲液反复洗涤3次，除去过量的聚电解质，得到表面带正电荷的第二微泡混悬液；
(4)向所述第二微泡混悬液中缓慢加入10mL制备例2所得的柠檬酸钠修饰的超顺磁性纳米颗粒的水溶液(其中超顺磁性纳米颗粒的量与第二微泡混悬液中脂质的摩尔比为0.18:1)，在350kW下超声振荡，吸附20min后，置于4℃冰箱中静置分层，弃下层清液；然后将上层液体用PBS缓冲液反复洗涤3次，得到磁性微泡超声造影剂，记为S2。
实施例3
按照以下步骤进行：
(1)将山梨醇酐单硬脂酸酯(脂质)、聚山梨醇酯80(脂质)以及泊洛沙姆F68(德国BASF，P21489-25G，表面活性A剂，HLB值＝29)按照1：1.2：1.65的摩尔比混合均匀，取10mL置于130℃灭菌锅中高温高压8min，之后连续搅拌冷却至40度；
(2)将步骤(1)所得物料与紫杉醇(化疗药物)以100:2的重量比混合；采用超声空化法，在室温下向所述混合所得物料中通入全氟化碳气体，在10kW的功率下超声空化3min，冷却至室温，形成第一微泡混悬液；
(3)将所述第一微泡混悬液静置12h后离心除去上层粒径较大的微泡，通过光镜测量微泡的粒径分布在0.8-3μm的范围内；然后向其中缓慢加入10mL聚乙烯亚胺溶液(浓度为0.8mg/L)，在350kW下超声振荡，吸附20min后，置于4℃冰箱中静置分层，弃下层清液；然后将上层液体用PBS缓冲液反复洗涤3次，除去过量的聚电解质，得到表面带正电荷的第二微泡混悬液；
(4)向所述第二微泡混悬液中缓慢加入10mL制备例3所得的柠檬酸钠修饰的超顺磁性纳米颗粒的水溶液(其中超顺磁性纳米颗粒的量与第二微泡混悬液中脂质的摩尔比为0.25:1)，在250kW下超声振荡，吸附40min后，置于4℃冰箱中静置分层，弃下层清液；然后将上层液体用PBS缓冲液反复洗涤3次，除去过量的超顺磁性纳米颗粒，得到磁性微泡超声造影剂，记为S3。
实施例4
参照实施例1的方法进行，所不同的是，改变两种脂质的配比，具体地，山梨醇酐单硬脂酸酯和聚山梨醇酯80加入的摩尔比为1：2.5。
最终得到磁性微泡超声造影剂，记为S4。
实施例5
参照实施例1的方法进行，所不同的是，将步骤(4)中所用的制备例1所得的柠檬酸钠修饰的超顺磁性纳米颗粒替换为等量的制备例4所得的柠檬酸钠修饰的超顺磁性纳米颗粒。
最终得到磁性微泡超声造影剂，记为S5。
实施例6
按照实施例1的方法进行，所不同的是，将泊洛沙姆替换为相同摩尔量的聚(异丁烯-马来酸酐)(西格玛奥德里奇，531278-250G)。
最终得到磁性微泡超声造影剂，记为S6。
实施例7
按照实施例1的方法进行，所不同的是，将泊洛沙姆替换为相同摩尔量的聚(马来酸酐-alt-1-十八碳烯)(西格玛奥德里奇，419117-250G)。
最终得到磁性微泡超声造影剂，记为S7。
实施例8
按照实施例1的方法进行，所不同的是，改变泊洛沙姆的投加量，具体地，按照山梨醇酐单硬脂酸酯：聚山梨醇酯80：泊洛沙姆F68＝1：1：1.2的摩尔比进行混合。
最终得到磁性微泡超声造影剂，记为S8。
实施例9
按照实施例1的方法进行，所不同的是，改变泊洛沙姆的投加量，具体地，按照山梨醇酐单硬脂酸酯：聚山梨醇酯80：泊洛沙姆F68＝1：1：1.0的摩尔比进行混合。
最终得到磁性微泡超声造影剂，记为S9。
实施例10
按照实施例1的方法进行，所不同的是，改变泊洛沙姆的投加量，具体地，按照山梨醇酐单硬脂酸酯：聚山梨醇酯80：泊洛沙姆F68＝1：1：0.8的摩尔比进行混合。
最终得到磁性微泡超声造影剂，记为S10。
实施例11
按照实施例1的方法进行，所不同的是，改变泊洛沙姆的投加量，具体地，按照山梨醇酐单硬脂酸酯：聚山梨醇酯80：泊洛沙姆F68＝1：1：0.6的摩尔比进行混合。
最终得到磁性微泡超声造影剂，记为S11。
实施例12
按照实施例1的方法进行，所不同的是，改变泊洛沙姆的投加量，具体地，按照山梨醇酐单硬脂酸酯：聚山梨醇酯80：泊洛沙姆F68＝1：1：2的摩尔比混合均匀。
最终得到磁性微泡超声造影剂，记为S12。
实施例13
按照实施例1的方法进行，所不同的是，将泊洛沙姆替换为相同摩尔量 的泊洛沙姆P123(西格玛奥德里奇，435465-250ML，表面活性A剂，HLB值＝9)。
最终得到磁性微泡超声造影剂，记为S13。
对比例1
按照实施例1的方法进行，所不同的是，不加入泊洛沙姆。
最终得到磁性微泡超声造影剂，记为D1。
对比例2
参照实施例1的方法进行，所不同的是，将实施例1所用的表面活性A剂泊洛沙姆替换为单硬脂酸甘油酯(天威泰达，RH30299-100g)。
最终得到磁性微泡超声造影剂，记为D2。
对比例3
参照实施例1的方法进行，所不同的是，将制备例1所得的柠檬酸钠修饰的超顺磁性纳米颗粒替换为相同量的对比制备例1所得的表面修饰有泊洛沙姆的超顺磁性Fe 3O 4纳米颗粒。
最终得到磁性微泡超声造影剂，记为D3。
对比例4
参照实施例1的方法进行，所不同的是，不加入柠檬酸钠修饰的超顺磁性纳米颗粒，即不进行步骤(3)和(4)，步骤(2)所得的表面带正电荷的第一微泡混悬液即为最终的微泡超声造影剂，记为D4。
测试例
(1)稳定性测试
超声造影剂在体内的稳定性通过超声造影剂的半衰期来反应，半衰期越 长，表明稳定性越高。具体的测试方法包括(以兔子为对象)：
在超声实时成像记录下，在注射超声造影剂后，随机选取超声图像内的一段血管区域，并记录该区域内的平均灰度值变化，达到灰度值最大值A的时间点记录t1，灰度值下降为A的50％的时间点记录为t2，则该种微泡的半衰期为|t1-t2|。注：要保证每次注射的超声造影剂浓度和剂量相同。
将实施例和对比例的超声造影剂所测得的半衰期结果分别记于表1中。
(2)机械指数测试
进行体外富集爆破实验，具体测试方法包括：
在一侧放置钕铁硼磁体(5000Gs)的纤维素软管(内径为1mm)中，分别注射相同浓度(10 6个/mL)的实施例和对比例的超声造影剂，并在生理流速下(100mL/h)进行磁性微泡的体外富集爆破实验。超声成像/爆破探头采用196阵元、中心频率为12MHz、频带宽度为6MHz的线阵探头，控制超声探头发出超声波的机械指数(控制范围为MI＝0.1-2.5，步进为0.1)，观察不同机械指数下微泡的爆破情况，并记录下各实施例和对比例的超声造影剂分别对应的爆破率大于90％时(时间小于10s)的(爆破率＝|爆破前-爆破后|/爆破前*100％)机械指数，将结果记于表1中。
(3)磁性测试
进行体外富集实验，具体测试方法包括：
在一侧放置钕铁硼磁体(5000Gs)的纤维素软管(内径为1mm)中，分别注射相同浓度(10 6个/mL)的实施例和对比例的超声造影剂，并在静止流速下(0mL/h)进行磁性微泡的体外富集爆破实验。超声成像探头采用196阵元、中心频率为12MHz、频带宽度为6MHz的线阵探头，控制超声探头发出超声波的机械指数(控制范围为MI<0.1)，观察不同机械指数下磁性微泡的磁性情况。
在15min内，若磁性微泡几乎全部富集在磁铁一侧(超声图像显示磁性微泡在磁铁端的灰度信号大于全部微泡信号的95％)，认为磁性正常；
在15min内，若磁性微泡半数以上富集在磁铁一侧(超声图像显示磁性微泡在磁铁端的灰度信号大于全部微泡信号的50％)，认为磁性弱；
在15min内，若磁性微泡分布均匀，无吸附效果(超声图像显示磁性微泡在磁铁端的灰度信号均匀分布在纤维素管中)，认为无磁性。
(4)以实施例1的磁性微泡超声波造影剂S1和对比例4的超声波造影剂D4为例，经过十次富集爆破试验，对S1和D4的富集灰度进行比较，分别如图2(b)和图2(a)所示。从图2可以明显看出，S1出现了明显的局部富集，而D4并未出现富集。
表1
  半衰期(min) 爆破机械指数MI 磁性
S1 6.3 1.1 正常
S2 5.8 1.1 正常
S3 5.6 1.0 正常
S4 5.3 0.9 正常
S5 5.6 1.0 较弱
S6 5.1 0.8 正常
S7 5.0 0.9 正常
S8 6.2 1 正常
S9 6 0.9 正常
S10 5.6 0.9 正常
S11 5.5 0.8 正常
S12 5.5 0.8 正常
S13 5.0 0.8 正常
D1 2 0.3 正常
D2 2.5 0.3 正常
D3 6.5 1.1 无磁性
D4 6.8 1.2 无磁性
从表1可以看出，本发明的超声造影剂组合物和超声造影剂能够兼顾更长的半衰期、更高的爆破机械指数并且有很好的磁性，从而使得磁性微泡超声造影剂满足体内循环靶向递送的要求。
并且从实施例1、8-11可以看出，当乳化剂的量在合适的范围时，乳化剂的含量越高，磁性造影剂半衰期越长且爆破机械指数阈值越高；从实施例12可以看出，当乳化剂含量过高时，磁性造影剂半衰期和爆破机械指数阈值会出现下降。因此本发明的超声造影剂的性能可控，对实际应用具有重要意义。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。

Claims (15)

  1. 一种磁性超声造影剂组合物，其特征在于，该磁性超声造影剂组合物包括脂质、表面活性剂和磁性纳米颗粒，其中，所述表面活性剂由表面活性A剂和表面活性B剂组成，所述表面活性A剂呈游离态，所述表面活性B剂呈结合态修饰于所述磁性纳米颗粒的表面；所述表面活性A剂为HLB值>8的非离子型表面活性剂，所述表面活性B剂为柠檬酸基化合物；相对于1mol的所述脂质，所述表面活性A剂的含量为0.1-1mol，所述表面活性B剂的含量为0.1-1mol，所述磁性纳米颗粒的含量为0.05-0.5mol。
  2. 根据权利要求1所述的磁性超声造影剂组合物，其中，所述表面活性A剂为HLB值>15的非离子型表面活性剂，优选为HLB值为20-35的非离子型表面活性剂；
    优选地，所述表面活性A剂选自泊洛沙姆、聚(异丁烯-马来酸酐)和聚(马来酸酐-alt-1-十八碳烯)中的一种或多种；
    优选地，所述表面活性A剂为泊洛沙姆，且所述表面活性B剂为柠檬酸盐。
  3. 根据权利要求1或2所述的磁性超声造影剂组合物，其中，相对于1mol的所述脂质，所述表面活性A剂的含量为0.2-0.6mol，所述表面活性B剂的含量为0.2-0.5mol，所述磁性纳米颗粒的含量为0.1-0.3mol。
  4. 根据权利要求1-3中任意一项所述的磁性超声造影剂组合物，其中，所述磁性纳米颗粒与所述表面活性B剂的摩尔比为1：(0.6-2.5)。
  5. 根据权利要求1-4中任意一项所述的磁性超声造影剂组合物，其中，所述超声造影剂组合物还包括药物，相对于100重量份的所述脂质、表面活 性剂和磁性纳米颗粒的重量之和，所述药物的含量为1-20重量份。
  6. 根据权利要求1-5中任意一项所述的磁性超声造影剂组合物，其中，所述脂质由山梨醇酐单硬脂酸酯和聚山梨醇酯80以1：(0.5-2)的摩尔比组合而成。
  7. 根据权利要求1-6中任意一项所述的磁性超声造影剂组合物，其中，所述磁性纳米颗粒的粒径为3-20nm的超顺磁性Fe 3O 4纳米颗粒。
  8. 一种磁性超声造影剂，其特征在于，该磁性超声造影剂中含有权利要求1-7中任意一项所述的磁性超声造影剂组合物，或者由所述磁性超声造影剂组合物制备得到。
  9. 一种制备磁性微泡超声造影剂的方法，其特征在于，所用原料包括权利要求1-7中任意一项所述的磁性超声造影剂组合物，所述方法包括以下步骤：
    (1)将脂质和表面活性A剂进行第一接触；
    (2)将药物与步骤(1)所得物料混合，向所述混合所得物料中通入气体，进行超声空化形成第一微泡混悬液；
    (3)将所述第一微泡混悬液进行负载正电荷化处理，得到表面带正电荷的第二微泡混悬液；
    (4)将表面修饰有表面活性B剂的磁性纳米颗粒与所述第二微泡混悬液进行第二接触。
  10. 根据权利要求9所述的方法，其中，在步骤(1)中，所述第一接触的条件包括：温度为110-130℃，时间为8-16min。
  11. 根据权利要求9或10所述的方法，其中，在步骤(2)中，所述混合在30-50℃的温度下进行。
  12. 根据权利要求9-11中任意一项所述的方法，其中，所述超声空化的条件包括：超声功率为8-12kW，时间为1-8min。
  13. 根据权利要求9-12中任意一项所述的方法，其中，在步骤(3)中，所述负载正电荷化处理的过程包括：将所述第一微泡混悬液与阳离子试剂水溶液进行接触；
    优选地，所述阳离子试剂水溶液与所述第一微泡混悬液的体积比为(0.8-1.2)：1，且所述阳离子试剂水溶液中的阳离子的浓度为0.5-2mg/L。
  14. 根据权利要求9-13中任意一项所述的方法，其中，所述方法还包括：将与阳离子试剂接触后的物料静置分层，取上层物料并用缓冲溶液进行洗涤。
  15. 根据权利要求9-14中任意一项所述的方法，其中，在步骤(4)中，所述第二接触的条件包括：对物料进行超声振荡，超声功率为170-420kW，时间为15-50min。
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CN113768543B (zh) * 2021-09-15 2024-03-22 南京超维景生物科技有限公司 超声造影成像方法与系统

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101711870A (zh) * 2009-11-23 2010-05-26 王黛芳 一种复合靶向给药系统的载体及其制备方法
CN103243073A (zh) * 2013-05-13 2013-08-14 东南大学 用于将纳米颗粒输入细胞的载体系统及方法
US20140161726A1 (en) * 2003-10-31 2014-06-12 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Reconstitutable Microsphere Compositions Useful As Ultrasonic Contrast Agents
CN104622848A (zh) * 2015-02-13 2015-05-20 西安交通大学 一种等离子体活化包膜微气泡
CN109045285A (zh) * 2018-11-01 2018-12-21 南京邮电大学 一种载药磁性微泡及其制备方法和应用
CN110575551A (zh) * 2018-06-08 2019-12-17 北京大学 一种超声造影剂及其制备方法
CN111729092A (zh) * 2020-06-28 2020-10-02 南京超维景生物科技有限公司 磁性超声造影剂组合物、磁性超声造影剂、磁性微泡超声造影剂及其制备方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2543296A1 (en) * 2003-10-31 2005-05-12 Point Biomedical Corporation Reconstitutable microsphere compositions useful as ultrasonic contrast agents

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140161726A1 (en) * 2003-10-31 2014-06-12 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Reconstitutable Microsphere Compositions Useful As Ultrasonic Contrast Agents
CN101711870A (zh) * 2009-11-23 2010-05-26 王黛芳 一种复合靶向给药系统的载体及其制备方法
CN103243073A (zh) * 2013-05-13 2013-08-14 东南大学 用于将纳米颗粒输入细胞的载体系统及方法
CN104622848A (zh) * 2015-02-13 2015-05-20 西安交通大学 一种等离子体活化包膜微气泡
CN110575551A (zh) * 2018-06-08 2019-12-17 北京大学 一种超声造影剂及其制备方法
CN109045285A (zh) * 2018-11-01 2018-12-21 南京邮电大学 一种载药磁性微泡及其制备方法和应用
CN111729092A (zh) * 2020-06-28 2020-10-02 南京超维景生物科技有限公司 磁性超声造影剂组合物、磁性超声造影剂、磁性微泡超声造影剂及其制备方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JIANG CHAOXUE ET AL.: "Determination of Hydrophilic-lipophilic Balance of Nonionic Emulsifier by Inverse Gas Chromatography", CHINA SYNTHETIC RUBBER INDUSTRY, vol. 14, no. 6, 1991, pages 399 - 401
See also references of EP4046659A4
SONG WEIXIANG, LUO YINDENG, ZHAO YAJING, LIU XINJIE, ZHAO JIANNONG, LUO JIE, ZHANG QUNXIA, RAN HAITAO, WANG ZHIGANG, GUO DAJING: "Magnetic nanobubbles with potential for targeted drug delivery and trimodal imaging in breast cancer: an in vitro study,", NANOMEDICINE, vol. 12, no. 9, 1 May 2017 (2017-05-01), pages 991 - 1009, XP009533334, ISSN: 1743-5889, DOI: 10.2217/nnm-2017-0027 *

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