技术领域Technical field
本发明涉及一种携载阿霉素的壳聚糖纳米级超声造影剂及其制备方法与应用，属于超声分子影像学技术领域。The invention relates to a chitosan nano-level ultrasonic contrast agent carrying doxorubicin and a preparation method and application thereof, and belongs to the technical field of ultrasonic molecular imaging.
背景技术Background technique
随着超声分子成像技术和生物纳米技术的迅猛发展，纳米级超声造影剂发展迅速。纳米级超声造影剂是分子影像学领域近年来的研究热点之一，也是靶向给药系统(Targeted drug delivery system,TDDS)的重要研究方向之一，其可以有效克服传统肿瘤药物治疗靶向性差、全身毒副作用大等缺点。与传统微米级超声造影剂(血池显像剂)不同，纳米级超声造影剂具有很强的穿透力，能穿过肿瘤组织血管壁间隙，其开发和应用还有利于进一步发展靶向性、高效性、小型化且具有辅助治疗作用的新型超声造影剂。纳米级超声造影剂是超声分子影像学发展的重要方向，也是肿瘤靶向性、可视化及精准治疗的新方向。With the rapid development of ultrasonic molecular imaging technology and biological nanotechnology, nano-scale ultrasound contrast agents have developed rapidly. Nano-scale ultrasound contrast agent is one of the research hotspots in the field of molecular imaging in recent years, and it is also one of the important research directions of Targeted drug delivery system (TDDS), which can effectively overcome the poor targeting of traditional tumor drug therapy. , systemic toxic side effects and other shortcomings. Unlike traditional micron-sized ultrasound contrast agents (blood pool imaging agents), nano-scale ultrasound contrast agents have a strong penetrating power and can pass through the vascular wall space of tumor tissues. Its development and application are also conducive to further development of targeting. A new type of ultrasound contrast agent that is highly efficient, miniaturized, and has an adjuvant therapeutic effect. Nano-scale ultrasound contrast agent is an important direction for the development of ultrasound molecular imaging, and it is also a new direction for tumor targeting, visualization and precision treatment.
壳膜材料是决定超声造影剂性能的关键要素。目前，多种材料被用于超声造影剂的制备，包括：脂质、高分子聚合体、表面活性剂等，但均存在一定的缺点。脂质超声造影剂稳定性良好，成像效果好，但价格较为昂贵并且对肾脏和肝脏都有潜在的毒性，存在安全性问题；高分子聚合体如乳酸/羟基乙酸共聚物(PLGA)等可制备出较为稳定的超声造影剂，延长诊断时间，但由于造影剂外壳较硬，弹性较差，成像较差，需要较高的超声输出频率，可能会对正常组织产生非治疗性损失；表面活性剂超声造影剂成像好，但稳定性差，不易被修饰。壳聚糖材料无毒性、无刺激性，不仅具有良好的生物相容性及生物可降解性，还具有独特的生理和药理活性，最重要的是它含有丰富的自由氨基和羟基等活性基团，具有良好的修饰性，是一种理想的超声造影剂壳膜材料。中国专利文献CN106139174A提供了一种包裹液态氟碳的基于壳聚糖衍生物纳米级超声造影剂的制备方法，是通过酰化反应对羧甲基壳聚糖进行改性，合成具有两亲性的正己酰羧甲基壳聚糖，在此基础上加入液态氟碳，采用超声乳化方法制得由液态氟碳内核和壳聚糖衍生物外壳构成的纳米级超声造影剂。该方法所用的高分子材料安全无毒，且造影剂制备工艺简便，操作条件温和。但是该方法制备的纳米级超声造影剂为纳米液滴状态，在实际应用时尚需一定的外界刺激使之发生液气相变，而且没有对其靶向性及载药特性作进一步研究及评价。Shell membrane material is a key element in determining the performance of ultrasound contrast agents. At present, a variety of materials are used for the preparation of ultrasound contrast agents, including: lipids, polymeric polymers, surfactants, etc., but all have certain disadvantages. Lipid ultrasound contrast agents have good stability and good imaging results, but they are expensive and potentially toxic to the kidneys and liver. There are safety problems. Polymers such as lactic acid/glycolic acid copolymer (PLGA) can be prepared. A more stable ultrasound contrast agent prolongs the diagnosis time, but because the contrast agent shell is hard, the elasticity is poor, the imaging is poor, and the high ultrasonic output frequency is required, which may cause non-therapeutic loss to normal tissues; surfactant Ultrasound contrast imaging is good, but its stability is poor and it is not easy to be modified. The chitosan material is non-toxic, non-irritating, not only has good biocompatibility and biodegradability, but also has unique physiological and pharmacological activities. Most importantly, it is rich in active groups such as free amino groups and hydroxyl groups. With good modification, it is an ideal ultrasound contrast agent shell membrane material. Chinese patent document CN106139174A provides a preparation method of a nanometer-based ultrasonic contrast agent based on chitosan derivative encapsulating liquid fluorocarbon, which is modified by acylation reaction to synthesize carboxymethyl chitosan, and has amphiphilic synthesis. The n-hexyl carboxymethyl chitosan was added with liquid fluorocarbon, and a nano-scale ultrasonic contrast agent composed of a liquid fluorocarbon core and a chitosan derivative shell was obtained by ultrasonic emulsification. The polymer material used in the method is safe and non-toxic, and the preparation process of the contrast agent is simple and the operating conditions are mild. However, the nano-scale ultrasound contrast agent prepared by the method is in the state of nano-droplet. In practical application, a certain external stimulus is required to cause liquid-gas phase change, and no further research and evaluation are made on its targeting and drug-loading characteristics.
发明内容Summary of the invention
针对现有技术的不足，本发明提供了一种携载阿霉素的壳聚糖纳米级超声造影剂及其制备方法，以壳聚糖为壳膜材料制备纳米级超声造影剂，该造影剂具有较强的增强显像能力，较高的药物包封率及载药量，生物安全性高。In view of the deficiencies of the prior art, the present invention provides a chitosan nano-grade ultrasonic contrast agent carrying doxorubicin and a preparation method thereof, and a nano-scale ultrasonic contrast agent prepared by using chitosan as a shell material, the contrast agent It has strong enhanced imaging ability, high drug encapsulation rate and drug loading, and high biosafety.
本发明还提供了上述携载阿霉素的壳聚糖纳米级超声造影剂在治疗肿瘤及制备抗肿瘤药物中的应用。The invention also provides the application of the above-mentioned doxorubicin-loaded chitosan nanometer ultrasound contrast agent in treating tumors and preparing antitumor drugs.
术语说明：Explanation of terms:
阿霉素：Doxorubicin(DOX)，是一种抗肿瘤抗生素，可抑制RNA和DNA的合成，对RNA的抑制作用最强，抗瘤谱较广，对多种肿瘤均有作用，属周期非特异性药物，对各种生长周期的肿瘤细胞都有杀灭作用。Doxorubicin (DOX), an anti-tumor antibiotic that inhibits the synthesis of RNA and DNA, has the strongest inhibitory effect on RNA, has a broad anti-tumor spectrum, and has a role in a variety of tumors. The drug has a killing effect on tumor cells of various growth cycles.
室温：25±2℃。Room temperature: 25 ± 2 ° C.
本发明的技术方案为：The technical solution of the present invention is:
一种携载阿霉素的壳聚糖纳米级超声造影剂，该纳米级超声造影剂以壳聚糖为壳膜，壳聚糖壳膜内部包裹有阿霉素和气态氟碳，所述纳米级超声造影剂的粒径为300～900nm，其中，壳聚糖和阿霉素的质量比为1:1～4。A chitosan nano-level ultrasound contrast agent carrying doxorubicin, the nano-scale ultrasound contrast agent adopts chitosan as a shell membrane, and the chitosan shell membrane is internally coated with doxorubicin and gaseous fluorocarbon, the nanometer The particle size of the ultrasonic contrast agent is 300-900 nm, wherein the mass ratio of chitosan to doxorubicin is 1:1 to 4.
上述携载阿霉素的壳聚糖纳米级超声造影剂的制备方法，步骤如下：The preparation method of the above-mentioned doxorubicin-loaded chitosan nano-level ultrasound contrast agent is as follows:
(1)将棕榈酸、卵磷脂分散于磷酸盐缓冲液中制成混悬液，向混悬液中加入壳聚糖及阿霉素，25℃共孵育20～40mim，得孵育产物；(1) Dispersing palmitic acid and lecithin in a phosphate buffer solution to prepare a suspension, adding chitosan and doxorubicin to the suspension, and incubating at 20 ° C for 20 to 40 mim to obtain an incubation product;
(2)向步骤(1)制得的孵育产物中充入气态氟碳，室温下超声震荡1～5min，得乳化产物；(2) The incubation product prepared in the step (1) is filled with gaseous fluorocarbon, and ultrasonically shaken at room temperature for 1 to 5 minutes to obtain an emulsified product;
(3)将步骤(2)制得的乳化产物于4℃静置，静置时间≥2h，分层后取下层液，稀释后低速离心，取下层液体，即为携载阿霉素的壳聚糖纳米级超声造影剂。(3) The emulsified product obtained in the step (2) is allowed to stand at 4 ° C, the standing time is ≥ 2 h, and the lower layer liquid is taken after stratification, and the mixture is diluted and centrifuged at a low speed to take the lower layer liquid, which is a shell carrying doxorubicin. A nanometer-scale ultrasound contrast agent.
根据本发明优选的，步骤(1)所述棕榈酸和卵磷脂的质量比为1:2，所述混悬液中棕榈酸的浓度为0.4～0.5g/L；其中棕榈酸纯度≥99％，购自sigma，产品编号200-312-9；卵磷脂纯度≥99％，购自sigma，产品编号232-715-0。According to the present invention, the mass ratio of palmitic acid to lecithin in step (1) is 1:2, the concentration of palmitic acid in the suspension is 0.4-0.5 g/L; wherein the purity of palmitic acid is ≥99%. , purchased from sigma, product number 200-312-9; lecithin purity ≥ 99%, purchased from sigma, product number 232-715-0.
根据本发明优选的，步骤(1)中所述磷酸盐缓冲液的组分为：Na2HPO4 8mM，KH2PO42mM，pH 7.2-7.4。According to a preferred embodiment of the invention, the phosphate buffer component of step (1) has a composition of: Na2 HPO4 8 mM, KH2 PO4 2 mM, pH 7.2-7.4.
根据本发明优选的，步骤(1)中所述壳聚糖和阿霉素的质量比为1:1～4，所述混悬液中壳聚糖的浓度为0.4～0.6g/L；其中壳聚糖的分子量为160kDa，购自sigma，产品编号MFCD00161512；所述阿霉素纯度≥98％，购自sigma，产品编号246-818-3。According to the preferred embodiment of the present invention, the mass ratio of chitosan to doxorubicin in step (1) is 1:1 to 4, and the concentration of chitosan in the suspension is 0.4 to 0.6 g/L; The molecular weight of chitosan was 160 kDa, purchased from sigma, product number MFCD00161512; the purity of doxorubicin was ≥98%, purchased from sigma, product number 246-818-3.
进一步优选的，所述壳聚糖和阿霉素的质量比为1:2，Further preferably, the mass ratio of the chitosan to the doxorubicin is 1:2,
根据本发明优选的，步骤(2)中所述气态氟碳为全氟丙烷气体。According to a preferred embodiment of the invention, the gaseous fluorocarbon in step (2) is a perfluoropropane gas.
根据本发明优选的，步骤(3)中所述稀释的倍数为5～10倍。According to a preferred embodiment of the invention, the dilution in step (3) is 5 to 10 times.
根据本发明优选的，步骤(3)中所述低速离心为500rpm离心5min。According to a preferred embodiment of the invention, the low speed centrifugation in step (3) is centrifuged at 500 rpm for 5 min.
上述携载阿霉素的壳聚糖纳米级超声造影剂联合超声靶向破坏微泡在治疗肿瘤中的应用。The above-mentioned doxorubicin-loaded chitosan nano-sound ultrasound contrast agent combined with ultrasonic targeted destruction of microbubbles in the treatment of tumors.
上述携载阿霉素的壳聚糖纳米级超声造影剂联合超声靶向破坏微泡在制备抗肿瘤药物中的应用。The above-mentioned doxorubicin-loaded chitosan nano-sound ultrasound contrast agent combined with ultrasonic targeted destruction of microbubbles for the preparation of antitumor drugs.
本发明的技术特点：棕榈酸和卵磷脂有助于壳聚糖的溶解，壳聚糖溶解后有利于孵育产物的制备，将壳聚糖与阿霉素的孵育产物采用超声乳化的方式，得到以壳聚糖为外壳、以阿霉素和气态氟碳为内核的乳化产物，经静置后，粒径较大的超声造影剂分布在上层，粒径相对较小的纳米级超声造影剂分布在下层，取下层液经稀释和低速离心，得到粒径较小且均匀的纳米级超声造影剂。The technical features of the invention: palmitic acid and lecithin contribute to the dissolution of chitosan, and the preparation of the incubation product is facilitated by the dissolution of chitosan, and the incubation product of chitosan and doxorubicin is obtained by phacoemulsification. An emulsion product with chitosan as the outer shell and doxorubicin and gaseous fluorocarbon as the core. After standing, the ultrasonic contrast agent with larger particle size is distributed in the upper layer, and the nano-sized ultrasound contrast agent with relatively small particle size is distributed. In the lower layer, the lower layer liquid was subjected to dilution and low-speed centrifugation to obtain a nano-sized ultrasonic contrast agent having a small particle size and uniformity.
有益效果：Beneficial effects:
1、本发明制备的携载阿霉素的壳聚糖纳米级超声造影剂，是以壳聚糖为壳膜，阿霉素(DOX)和全氟丙烷气体包裹在壳聚糖壳膜内部，粒径为300～900nm，平均粒径为641nm(聚合物分散指数PDI：0.256)，在纳米级范围，能穿过肿瘤组织血管壁间隙，对于肿瘤的治疗具有靶向性和高效性。1. The doxorubicin-loaded chitosan nano-sound ultrasound contrast agent prepared by the invention is prepared by chitosan as a shell membrane, and doxorubicin (DOX) and perfluoropropane gas are encapsulated in the chitosan shell membrane. The particle size is 300-900 nm, and the average particle size is 641 nm (polymer dispersion index PDI: 0.256). In the nanometer range, it can pass through the blood vessel wall gap of the tumor tissue, and is targeted and highly effective for the treatment of tumors.
2、本发明制备的携载阿霉素的壳聚糖纳米级超声造影剂具有较强的增强显像能力，而且可以在较长的时间内维持显影，还具有较高的药物包封率及载药量，生物安全性高，辐照强度为0.5W/cm2，辐照时间为30sec或60sec，细胞生存率均＞80％，适宜于超声显影及肿瘤疾病的辅助治疗。2. The doxorubicin-loaded chitosan nano-sound ultrasound contrast agent prepared by the invention has strong enhanced imaging ability, and can maintain development for a long time, and has a high drug encapsulation rate and The drug loading, biosafety, irradiation intensity is 0.5W/cm2 , irradiation time is 30sec or 60sec, cell survival rate is >80%, suitable for ultrasound imaging and adjuvant treatment of tumor diseases.
3、本发明利用超声辐照的方式，一方面可以增强携载阿霉素的壳聚糖纳米级超声造影剂显影，另一方面可以借助超声辐照产生的声孔效应促进阿霉素更多的进入肿瘤细胞内，实现诊疗一体化。3. The invention utilizes the method of ultrasonic irradiation to enhance the development of chitosan nano-sound ultrasound contrast agent carrying doxorubicin on the one hand, and promote the doxorubicin by the sonic hole effect generated by ultrasonic irradiation on the other hand. Into the tumor cells, to achieve integration of diagnosis and treatment.
4、本发明制备的携载DOX的壳聚糖纳米级超声造影剂联合超声靶向破坏微泡(UTMD)能显著提高DOX的提取率和降低肿瘤细胞生存率，有利于肿瘤疾病的治疗。4. The DOX-loaded chitosan nano-scale ultrasound contrast agent prepared by the invention combined with ultrasonic targeted destruction microbubbles (UTMD) can significantly improve the extraction rate of DOX and reduce the survival rate of tumor cells, and is beneficial to the treatment of tumor diseases.
附图说明DRAWINGS
图1是携载DOX的壳聚糖纳米级超声造影剂的光镜图；Figure 1 is a photomicrograph of a chitosan nano-scale ultrasound contrast agent carrying DOX;
图2是携载DOX的壳聚糖纳米级超声造影剂的粒径图；2 is a particle size diagram of a DOX-loaded chitosan nano-scale ultrasound contrast agent;
图3是体外自制超声增强显像装置；Figure 3 is an in vitro self-made ultrasound enhanced imaging device;
图4是携载DOX的壳聚糖纳米级超声造影剂体外显影图；Figure 4 is an in vitro development of a chitosan nanoscale ultrasound contrast agent carrying DOX;
图5是携载DOX的壳聚糖纳米级超声造影剂体外显影随时间变化趋势图；Figure 5 is a graph showing the trend of in vitro development of a chitosan nano-sized ultrasound contrast agent carrying DOX over time;
图6是不同超声参数下携载DOX的壳聚糖纳米级超声造影剂的生物安全性评价；Figure 6 is a biosafety evaluation of chitosan nanoscale ultrasound contrast agent carrying DOX under different ultrasound parameters;
图7是携载DOX的壳聚糖纳米级超声造影剂中DOX摄取情况的流式细胞术图；Figure 7 is a flow cytometry diagram of DOX uptake in a chitosan nanoscale ultrasound contrast agent carrying DOX;
图8是携载DOX的壳聚糖纳米级超声造影剂对乳腺癌MCF-7细胞生存率影响图。Figure 8 is a graph showing the effect of DOX-loaded chitosan nano-grade ultrasound contrast agent on the survival rate of breast cancer MCF-7 cells.
具体实施方式Detailed ways
下面结合实施例对本发明做进一步的说明，但本发明的保护范围并不仅限于此。The present invention will be further described below in conjunction with the embodiments, but the scope of protection of the present invention is not limited thereto.
棕榈酸购自sigma，产品编号200-312-9；卵磷脂购自sigma，产品编号232-715-0；壳聚糖购自sigma，产品编号MFCD00161512；阿霉素购自sigma，产品编号246-818-3。Palmitic acid was purchased from sigma, product number 200-312-9; lecithin was purchased from sigma, product number 232-715-0; chitosan was purchased from sigma, product number MFCD00161512; doxorubicin was purchased from sigma, product number 246- 818-3.
本实施例涉及药品及试剂如无特殊说明，均为普通市售产品。The present embodiment relates to drugs and reagents which are all commercially available products unless otherwise specified.
实施例1：携载DOX的壳聚糖纳米级超声造影剂的制备Example 1: Preparation of DOX-loaded chitosan nano-scale ultrasound contrast agent
一种携载阿霉素的壳聚糖纳米级超声造影剂的制备方法，步骤如下：A preparation method of chitosan nanometer ultrasonic contrast agent carrying doxorubicin, the steps are as follows:
(1)称取棕榈酸和卵磷脂分散于磷酸盐缓冲液(Na2HPO4 8mM，KH2PO4 2mM，pH7.2)中，混匀制成混悬液备用，所述混悬液中，棕榈酸的浓度为0.45g/L，卵磷脂的浓度为0.9g/L；向上述混悬液中加入壳聚糖(分子量为160kDa)及阿霉素，壳聚糖及阿霉素在混悬液中的浓度分别为0.5g/L和1g/L，25℃共孵育30mim，得孵育产物；(1) Weigh palmitic acid and lecithin dispersed in phosphate buffer (Na2 HPO4 8 mM, KH2 PO4 2 mM, pH 7.2), and mix to prepare a suspension for use in the suspension. , the concentration of palmitic acid is 0.45g / L, the concentration of lecithin is 0.9g / L; chitosan (molecular weight 160kDa) and doxorubicin, chitosan and doxorubicin are mixed into the above suspension. The concentration in the suspension was 0.5 g/L and 1 g/L, respectively, and the mixture was incubated at 25 ° C for 30 mim to obtain an incubation product;
(2)向步骤(1)制得的孵育产物中充入全氟丙烷气体，室温下超声震荡2min，得乳化产物；(2) The incubation product prepared in the step (1) is filled with perfluoropropane gas, and ultrasonically shaken at room temperature for 2 minutes to obtain an emulsified product;
(3)将步骤(2)制得的乳化产物置于4℃静置2h，分层后取下层液，稀释5倍后500rpm低速离心5min，取下层液体，即为携载阿霉素的壳聚糖纳米级超声造影剂(DOX-NB)。(3) The emulsified product obtained in the step (2) is allowed to stand at 4 ° C for 2 h, and the lower layer liquid is taken after stratification, diluted 5 times, and then centrifuged at 500 rpm for 5 min at low speed to take the lower layer liquid, which is a shell carrying doxorubicin. A nanometer-scale ultrasound contrast agent (DOX-NB).
取上述制备的携载阿霉素的壳聚糖纳米级超声造影剂，稀释后滴加到载玻片上，通过光学显微镜观察造影剂的表观形貌，结果如图1所示，1000×光学显微镜下可见造影剂均呈球形，粒径均一，分散均匀无聚集；The doxorubicin-loaded chitosan nano-sized ultrasound contrast agent prepared above was diluted and added to a glass slide, and the apparent morphology of the contrast agent was observed by an optical microscope. The result is shown in Fig. 1, 1000× optical. Under the microscope, the contrast agents were all spherical, the particle size was uniform, and the dispersion was uniform without aggregation.
取上述制备的携载阿霉素的壳聚糖纳米级超声造影剂，稀释后，使用纳米激光粒度及Zeta电位分析仪检测超声造影剂的粒径，结果如图2所示，造影剂的粒径为300～900nm，造影剂的平均粒径为641nm(聚合物分散指数PDI：0.256)，表面电位为67.12±2.1mW。The doxorubicin-loaded chitosan nano-scale ultrasound contrast agent prepared above is diluted, and the particle size of the ultrasonic contrast agent is detected by using a nano-laser particle size and a zeta potential analyzer. The result is shown in FIG. The diameter was 300 to 900 nm, the average particle diameter of the contrast agent was 641 nm (polymer dispersion index PDI: 0.256), and the surface potential was 67.12 ± 2.1 mW.
实施例2Example 2
一种携载阿霉素的壳聚糖纳米级超声造影剂的制备方法，步骤如下：A preparation method of chitosan nanometer ultrasonic contrast agent carrying doxorubicin, the steps are as follows:
(1)称取棕榈酸和卵磷脂分散于磷酸盐缓冲液(Na2HPO4 8mM，KH2PO4 2mM，pH7.3)中，混匀制成混悬液备用，所述混悬液中，棕榈酸的浓度为0.4g/L，卵磷脂的浓度为0.8g/L；向上述混悬液中加入壳聚糖(分子量为160kDa)及阿霉素，壳聚糖及阿霉素在混悬液中的浓度分别为0.4g/L和1.6g/L，25℃共孵育25mim，得孵育产物；(1) Weigh palmitic acid and lecithin dispersed in phosphate buffer (Na2 HPO4 8 mM, KH2 PO4 2 mM, pH 7.3), and mix to prepare a suspension for use in the suspension. , the concentration of palmitic acid is 0.4g / L, the concentration of lecithin is 0.8g / L; chitosan (molecular weight 160kDa) and doxorubicin, chitosan and doxorubicin are mixed into the above suspension. The concentration in the suspension was 0.4 g/L and 1.6 g/L, respectively, and 25 μm was incubated at 25 ° C to obtain an incubation product;
(2)向步骤(1)制得的孵育产物中充入全氟丙烷气体，室温下超声震荡3min，得乳化产物；(2) The incubation product prepared in the step (1) is filled with perfluoropropane gas, and ultrasonically shaken at room temperature for 3 minutes to obtain an emulsified product;
(3)将步骤(2)制得的乳化产物置于4℃静置4h，分层后取下层液，稀释6倍后500rpm低速离心5min，取下层液体，即为携载阿霉素的壳聚糖纳米级超声造影剂(DOX-NB)。(3) The emulsified product obtained in the step (2) is allowed to stand at 4 ° C for 4 h, and the lower layer liquid is taken after stratification, diluted 6 times, and then centrifuged at 500 rpm for 5 min at low speed to take the lower layer liquid, which is a shell carrying doxorubicin. A nanometer-scale ultrasound contrast agent (DOX-NB).
本实施例制备的携载阿霉素的壳聚糖纳米级超声造影剂在1000×光学显微镜下呈球形，粒径均一，分散均匀无聚集，造影剂的粒径为300～900nm。The doxorubicin-loaded chitosan nano-sound ultrasound contrast agent prepared in this embodiment has a spherical shape under a 1000× optical microscope, uniform particle size, uniform dispersion and no aggregation, and the particle size of the contrast agent is 300-900 nm.
实施例3Example 3
一种携载阿霉素的壳聚糖纳米级超声造影剂的制备方法，步骤如下：A preparation method of chitosan nanometer ultrasonic contrast agent carrying doxorubicin, the steps are as follows:
(1)称取棕榈酸和卵磷脂分散于磷酸盐缓冲液(Na2HPO4 8mM，KH2PO4 2mM，pH7.4)中，混匀制成混悬液备用，所述混悬液中，棕榈酸的浓度为0.5g/L，卵磷脂的浓度为1.0g/L；向上述混悬液中加入壳聚糖(分子量为160kDa)及阿霉素，壳聚糖及阿霉素在混悬液中的浓度分别为0.6g/L和1.8g/L，25℃共孵育35mim，得孵育产物；(1) Weigh palmitic acid and lecithin dispersed in phosphate buffer (Na2 HPO4 8 mM, KH2 PO4 2 mM, pH 7.4), and mix to prepare a suspension for use in the suspension. The concentration of palmitic acid is 0.5g/L, and the concentration of lecithin is 1.0g/L; chitosan (molecular weight 160kDa) and doxorubicin are added to the above suspension, and chitosan and doxorubicin are mixed. The concentrations in the suspension were 0.6 g/L and 1.8 g/L, respectively, and the mixture was incubated at 25 ° C for 35 mim to obtain the incubation product;
(2)向步骤(1)制得的孵育产物中充入全氟丙烷气体，室温下超声震荡4min，得乳化产物；(2) The incubation product prepared in the step (1) is filled with perfluoropropane gas, and ultrasonically shaken at room temperature for 4 minutes to obtain an emulsified product;
(3)将步骤(2)制得的乳化产物置于4℃静置6h，分层后取下层液，稀释8倍后500rpm低速离心5min，取下层液体，即为携载阿霉素的壳聚糖纳米级超声造影剂(DOX-NB)。(3) The emulsified product obtained in the step (2) is allowed to stand at 4 ° C for 6 h, and the lower layer liquid is taken after stratification, diluted 8 times, and then centrifuged at 500 rpm for 5 min at low speed to take the lower layer liquid, which is a shell carrying doxorubicin. A nanometer-scale ultrasound contrast agent (DOX-NB).
本实施例制备的携载阿霉素的壳聚糖纳米级超声造影剂在1000×光学显微镜下呈球形，粒径均一，分散均匀无聚集，造影剂的粒径为300～900nm。The doxorubicin-loaded chitosan nano-sound ultrasound contrast agent prepared in this embodiment has a spherical shape under a 1000× optical microscope, uniform particle size, uniform dispersion and no aggregation, and the particle size of the contrast agent is 300-900 nm.
实施例4：DOX-NB的药物包封率及载药量检测Example 4: Drug Encapsulation Rate and Drug Loading of DOX-NB
取实施例1～3在制备DOX-NB时静置后的下层液，采用酶标仪检测下层液中底层液体在480nm处的吸光度，根据标准曲线计算底层液体中游离的DOX质量，再根据以下公式计算DOX-NB的药物包封率(EE％)和载药量(LD％)：Taking the lower liquid after standing in the preparation of DOX-NB in Examples 1 to 3, the absorbance of the bottom liquid in the lower liquid at 480 nm was detected by a microplate reader, and the free DOX mass in the bottom liquid was calculated according to the standard curve, and then according to the following The formula calculates the drug encapsulation efficiency (EE%) and drug loading (LD%) of DOX-NB:
EE％＝(载有的DOX质量/加入的初始DOX质量)×100％。EE% = (loaded DOX quality / initial DOX quality added) x 100%.
LD％＝(载有的DOX质量/加入的壳聚糖纳米级超声造影剂的质量)×100％LD%=(quality of DOX loaded/mass of added chitosan nano-sound ultrasound contrast agent)×100%
其中，among them,
载有的DOX质量＝加入的初始DOX质量-游离的DOX质量，DOX quality carried = initial DOX quality added - free DOX mass,
加入的壳聚糖纳米级超声造影剂的质量为加入的初始原料的总质量，包括壳聚糖、阿霉素、棕榈酸和卵磷脂。The mass of chitosan nano-scale ultrasound contrast agent added is the total mass of the starting materials added, including chitosan, doxorubicin, palmitic acid and lecithin.
计算结果如表1所示，本发明制备的DOX-NB的药物包封率在50％以上，载药量≥59.38mg DOX/g造影剂。The calculation results are shown in Table 1. The DOX-NB prepared by the present invention has a drug encapsulation efficiency of 50% or more and a drug loading amount of ≥59.38 mg of DOX/g contrast agent.
表1不同携载DOX的壳聚糖超声纳米级造影剂的包封率及载药量Table 1 Encapsulation efficiency and drug loading of different chitosan ultrasonic nano-scale contrast agents carrying DOX
实施例5：DOX-NB的体外超声显影能力检测Example 5: In vitro ultrasonic development capability detection of DOX-NB
自制4％琼脂凝胶模型，观察实施例1制备的DOX-NB的增强显像能力，操作装置如图3所示，将浓度为1.0×106bubbles/mL的DOX-NB加入琼脂凝胶模型的圆孔中，采用临床诊断超声仪(LOGIQ E9；GE，USA)检测超声显影能力，主要参数为：频率9.0MHz；机械指数：0.12；焦距：3.0cm；动态范围：60dB，二维与超声造影模式同步观察，参数设置保持不变。The 4% agar gel model was prepared, and the enhanced imaging ability of DOX-NB prepared in Example 1 was observed. The operation device was as shown in Fig. 3. DOX-NB at a concentration of 1.0 × 106 bubbles/mL was added to the agar gel model. In the circular hole, the ultrasonic imaging capability was measured by a clinical diagnostic ultrasound system (LOGIQ E9; GE, USA). The main parameters were: frequency 9.0 MHz; mechanical index: 0.12; focal length: 3.0 cm; dynamic range: 60 dB, two-dimensional and ultrasound The contrast mode is observed synchronously and the parameter settings remain unchanged.
用超声仪内部工作站存储图像资料，图像如图4所示，在灰阶显像和增强显像模式下，均能呈现出清晰的图像；Image J软件分析获取图像的灰度值，结果如图5所示，以上结果都表明，该造影剂有较强的超声显影能力，并能在较长时间(15min)内维持显影。The image data is stored by the internal workstation of the ultrasound system. The image is shown in Figure 4. In the gray-scale imaging and enhanced imaging mode, the image can be displayed clearly; the image J software analyzes the gray value of the image, and the result is shown in the figure. As shown in Fig. 5, the above results indicate that the contrast agent has strong ultrasonic development ability and can maintain development for a long time (15 min).
实施例6：壳聚糖纳米级超声造影剂的生物安全性检测Example 6: Biosafety detection of chitosan nanometer ultrasound contrast agent
按照实施例1所述的制备方法制备壳聚糖纳米级超声造影剂，不同之处在于步骤(1)中不添加阿霉素。取上述制备的壳聚糖纳米级超声造影剂，使用CCK-8试剂测定其在MCF-7细胞系中的生物安全性，具体操作为：将MCF-7细胞接种于96孔板中，以含10％FBS的DMEM为培养基，细胞的密度为1.0×104个/孔，37℃、5％CO2孵育箱中培养24h，细胞贴壁，将培养基更换为含有上述壳聚糖纳米级超声造影剂的新鲜10％FBS DMEM培养基，DMEM培养基中壳聚糖纳米级超声造影剂的体积分数分别为0、10％、20％、30％，并进行超声辐照，超声辐照处理如下表所示：The chitosan nano-sound ultrasound contrast agent was prepared according to the preparation method described in Example 1, except that no doxorubicin was added in the step (1). The chitosan nano-scale ultrasound contrast agent prepared above was used to determine the biosafety of the MCF-7 cell line using CCK-8 reagent. The specific operation was as follows: MCF-7 cells were seeded in a 96-well plate to contain 10% FBS DMEM was used as the medium, the density of the cells was 1.0×104 cells/well, cultured in a 37° C., 5% CO2 incubator for 24 hours, the cells were attached, and the medium was changed to contain the above chitosan nanoscale. Ultrasound contrast agent in fresh 10% FBS DMEM medium, volume fraction of chitosan nanometer ultrasound contrast agent in DMEM medium was 0, 10%, 20%, 30%, respectively, and ultrasonic irradiation, ultrasonic irradiation treatment As shown in the following table:
表2不同的超声辐照处理方式Table 2 different ultrasonic irradiation treatment methods
辐照结束后，37℃、5％CO2孵育箱中培养48h，然后用PBS洗涤细胞，加入含有10μLCCK-8试剂的新鲜10％FBS DMEM培养基，继续孵育1-4h，用酶标仪在480nm波长处检测其光吸收值。结果如图6所示：当壳聚糖纳米级超声造影剂的体积分数≤30％，辐照强度为0.5W/cm2时，各组细胞生存率均＞80％；当将辐照强度提高至1.0W/cm2时，壳聚糖纳米级超声造影剂的体积分数≤20％，细胞生存率也在80％以上，表明在一定的超声强度范围之内，该造影剂细胞毒性小，生物安全性好。After the irradiation, the cells were cultured in a 37 ° C, 5% CO2 incubator for 48 h, then the cells were washed with PBS, and fresh 10% FBS DMEM medium containing 10 μL of LCCK-8 reagent was added, and incubation was continued for 1-4 h, using a microplate reader. The light absorption value was measured at a wavelength of 480 nm. The results are shown in Fig. 6. When the volume fraction of chitosan nano-scale ultrasound contrast agent is ≤30% and the irradiation intensity is 0.5W/cm2 , the survival rate of each group is >80%; when the irradiation intensity is increased At 1.0W/cm2 , the volume fraction of chitosan nano-grade ultrasound contrast agent is ≤20%, and the cell survival rate is also above 80%, indicating that the contrast agent has little cytotoxicity within a certain range of ultrasonic intensity. Good security.
实施例7：DOX-NB联合靶向破坏微泡(UTMD)对DOX摂取率的影响Example 7: Effect of DOX-NB combined targeted destruction microbubbles (UTMD) on DOX extraction rate
取实施例1制备的DOX-NB，检测MCF-7细胞对DOX-NB中DOX的摄取率，步骤如下：将MCF-7细胞以每孔2.5×105个细胞的密度接种在6孔板中过夜贴壁，更换新鲜的10％FBSDMEM培养基，10％FBS DMEM培养基中分别含有游离DOX、DOX-NB、游离DOX和UTMD、DOX-NB和UTMD，其中，DOX-NB在培养基中的体积分数为20％，各组DOX的含量保持一致。37℃、5％CO2孵育箱中培养1h，给予超声辐照或无超声辐照处理，处理方式如下表：The DOX-NB prepared in Example 1 was used to detect the DOX uptake rate of MCF-7 cells in DOX-NB. The procedure was as follows: MCF-7 cells were seeded in a 6-well plate at a density of 2.5×105 cells per well. Overnight, add fresh 10% FBSDMEM medium, 10% FBS DMEM medium containing free DOX, DOX-NB, free DOX and UTMD, DOX-NB and UTMD, respectively, DOX-NB in the medium The volume fraction was 20%, and the content of DOX in each group remained the same. Incubate for 1 h at 37 ° C, 5% CO2 incubator, and give ultrasonic irradiation or no ultrasonic irradiation. The treatment methods are as follows:
表3.包含不同成分的DMEM培养基中不同的超声辐照处理方式Table 3. Different ultrasonic irradiation treatments in DMEM medium containing different components
超声辐照处理结束后，收集MCF-7细胞进行流式细胞术检测，结果如图7所示，结果表明：DOX-NB联合UTMD组DOX摄取效率显著高于对照组和游离DOX联合UTMD组，且辐照时间60sec时的摄取效率高于辐照时间30sec时的摄取效率。After the end of the ultrasonic irradiation treatment, MCF-7 cells were collected for flow cytometry. The results are shown in Figure 7. The results showed that the DOX uptake efficiency of DOX-NB combined with UTMD group was significantly higher than that of the control group and free DOX combined with UTMD group. Moreover, the uptake efficiency at the irradiation time of 60 sec was higher than the uptake efficiency at the irradiation time of 30 sec.
实施例8：DOX-NB联合靶向破坏微泡(UTMD)对MCF-7细胞的作用Example 8: Effect of DOX-NB combined with targeted disruption of microvesicles (UTMD) on MCF-7 cells
取实施例1制备的DOX-NB，使用CCK-8试剂测定其联合UTMD对MCF-7细胞的作用，预测DOX-NB在肿瘤治疗方面的应用价值。具体操作为：将MCF-7细胞接种于96孔板中，以含10％FBS的DMEM为培养基，细胞的密度为1.0×104个/孔，37℃、5％CO2孵育箱中过夜培养，细胞贴壁，更换新鲜的10％FBS DMEM培养基，培养基中分别含有游离DOX、DOX-NB、游离DOX和UTMD、DOX-NB和UTMD，其中，DOX-NB在培养基中的体积分数为20％，各组DOX的含量保持一致，给予超声辐照或无超声辐照处理，处理方式如表3所示；另外，以不含有其他成分的10％FBS DMEM培养基培养的MCF-7细胞为空白对照，无超声辐照处理。The DOX-NB prepared in Example 1 was used to determine the effect of combined UTMD on MCF-7 cells using CCK-8 reagent, and the application value of DOX-NB in tumor therapy was predicted. The specific operation was as follows: MCF-7 cells were seeded in a 96-well plate, and DMEM containing 10% FBS was used as a medium. The density of the cells was 1.0×104 cells/well, and the cells were incubated overnight at 37° C. in a 5% CO2 incubator. Culture, cell adherence, replacement of fresh 10% FBS DMEM medium containing free DOX, DOX-NB, free DOX and UTMD, DOX-NB and UTMD, respectively, wherein the volume of DOX-NB in the medium The score was 20%. The content of DOX in each group was consistent. Ultrasonic irradiation or no ultrasonic irradiation was applied. The treatment was as shown in Table 3. In addition, MCF-cultured in 10% FBS DMEM medium containing no other ingredients. 7 cells were blank controls and were not treated with ultrasound.
超声辐照结束后，37℃、5％CO2孵育箱中培养24小时，然后用PBS洗涤细胞，加入含有10μLCCK-8试剂的新鲜10％FBS DMEM培养基，继续孵育1-4h，用酶标仪在480nm波长处检测其光吸收值，计算细胞生存率，结果如图8所示，结果显示：DOX-NB联合UTMD在超声强度为0.5W/cm2、辐照时间为60sec时，MCF-7细胞的生存率最低，为2.2±0.9％，明显低于DOX-NB组(21.0±2.2％)和游离DOX组(6.4±0.7％)，差异均有统计学差异(p＜0.01)，表明DOX-NB联合UTMD在超声辐照的条件下可有效降低肿瘤细胞的生存率，应用于肿瘤治疗。After the end of the ultrasonic irradiation, culture in a 37 ° C, 5% CO2 incubator for 24 hours, then wash the cells with PBS, add fresh 10% FBS DMEM medium containing 10μLCCK-8 reagent, continue to incubate for 1-4h, using enzyme label The instrument absorbs the light absorption value at 480 nm and calculates the cell survival rate. The results are shown in Fig. 8. The results show that DOX-NB combined with UTMD has an ultrasonic intensity of 0.5 W/cm2 and an irradiation time of 60 sec, MCF- The survival rate of the 7 cells was the lowest, 2.2±0.9%, which was significantly lower than that of the DOX-NB group (21.0±2.2%) and the free DOX group (6.4±0.7%). The difference was statistically significant (p<0.01). DOX-NB combined with UTMD can effectively reduce the survival rate of tumor cells under ultrasound irradiation conditions and is applied to tumor treatment.
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| CN201811219930.0ACN109260480B (en) | 2018-10-19 | 2018-10-19 | A kind of preparation method of chitosan nanometer ultrasonic contrast agent carrying adriamycin |
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| CN115252829A (en)* | 2022-08-07 | 2022-11-01 | 山西省人民医院 | A kind of preparation method of ultrasound contrast agent capable of targeting renal cancer administration |
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| CN109260480B (en) | 2019-06-18 |
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| TR01 | Transfer of patent right | Effective date of registration:20250121 Address after:361000 building 6, 2010 wengjiao West Road (Xiamen biomedical industrial park), Haicang District, Xiamen City, Fujian Province Patentee after:Lipin Pharmaceutical (Xiamen) Co.,Ltd. Country or region after:China Address before:250012 No. 107 west Wenhua Road, Lixia District, Shandong, Ji'nan Patentee before:QILU HOSPITAL OF SHANDONG University Country or region before:China |