CN112263679B - A targeted oxygen-carrying nano-sound sensitizer and its preparation method - Google Patents
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Abstract
Description
技术领域Technical field
本发明涉及纳米医学领域,特别涉及一种靶向载氧纳米声敏剂及其制备方法。The invention relates to the field of nanomedicine, and in particular to a targeted oxygen-carrying nanometer sonosensitizer and a preparation method thereof.
背景技术Background technique
现有技术中,肿瘤光学治疗因具有非侵入和时空可控的优势特征而逐渐成为癌症治疗的研究热点。肿瘤光学治疗主要包括光动力治疗(PDT)和光热治疗(PTT)。然而,近红外光组织穿透深度有限,深层肿瘤的治疗效果不佳。与之相比,声动力治疗(SDT)吸引了人们的广泛关注,利用超声更强的组织强穿透能力,使聚集在深层肿瘤中的声敏剂产生活性氧(ROS)或空化效应来治疗肿瘤。目前,声动力治疗已用于临床,结合内分泌疗法和免疫疗法成功治愈了3例晚期乳腺癌患者。常用的声敏剂分子包括:卟啉、卟啉衍生物、抗癌药物DOX、酸性黄、亚甲基蓝、多羟基富勒烯等有机分子。然而,这些传统有机声敏剂分子存在生物利用度低、氧依赖以及缺乏肿瘤靶向性等问题,导致声动力治疗效率低,严重阻碍该方法的临床推广。Among existing technologies, tumor optical therapy has gradually become a research hotspot in cancer treatment due to its advantages of non-invasiveness and spatiotemporal controllability. Optical tumor treatment mainly includes photodynamic therapy (PDT) and photothermal therapy (PTT). However, the tissue penetration depth of near-infrared light is limited, and the treatment effect of deep-seated tumors is poor. In contrast, sonodynamic therapy (SDT) has attracted widespread attention. It uses the stronger tissue penetration ability of ultrasound to cause the sonosensitizer accumulated in deep tumors to produce reactive oxygen species (ROS) or cavitation effect. Treat tumors. Currently, sonodynamic therapy has been used in clinical practice, and three patients with advanced breast cancer were successfully cured by combining endocrine therapy and immunotherapy. Commonly used sonosensitizer molecules include: porphyrin, porphyrin derivatives, anti-cancer drug DOX, acid yellow, methylene blue, polyhydroxyfullerene and other organic molecules. However, these traditional organic sonosensitizer molecules have problems such as low bioavailability, oxygen dependence, and lack of tumor targeting, resulting in low sonodynamic therapy efficiency and seriously hindering the clinical promotion of this method.
近年来,全球纳米技术的迅猛发展给癌症治疗带来一场全新的变革。纳米材料的新型药物载体可实现特异的靶向定位,将药物高效地富集到肿瘤病灶;同时通过调控纳米颗粒的尺寸、形貌、表面电荷及配体修饰等理化性质,突破肿瘤内部胶原、粘多糖、蛋白聚糖等构成的高密度间隙基质屏障,将荷载的药物递送到肿瘤内部,从而增强抗肿瘤的疗效。针对传统声敏剂存在生物利用度低、靶向性差、氧依赖等瓶颈问题,纳米技术提供了新策略和新方向,为新型纳米声动力药物制剂的临床推广带来了新的机遇。发展生物利用度高、靶向性好和改善肿瘤缺氧微环境的新型纳米声敏剂已成为现今声动力治疗领域的重要研究方向。In recent years, the rapid development of global nanotechnology has brought a new revolution to cancer treatment. New drug carriers based on nanomaterials can achieve specific targeting and efficiently concentrate drugs into tumor lesions; at the same time, by regulating the size, morphology, surface charge, ligand modification and other physical and chemical properties of nanoparticles, they can break through the internal collagen, The high-density interstitial matrix barrier composed of mucopolysaccharides, proteoglycans, etc., delivers the loaded drugs into the tumor, thus enhancing the anti-tumor efficacy. In view of the bottleneck problems of traditional sonosensitizers such as low bioavailability, poor targeting, and oxygen dependence, nanotechnology provides new strategies and directions, and brings new opportunities for the clinical promotion of new nanosonodynamic pharmaceutical preparations. The development of new nano-sound sensitizers with high bioavailability, good targeting and improving the hypoxic microenvironment of tumors has become an important research direction in the field of sonodynamic therapy today.
但目前的纳米声敏剂,制备工艺复杂,无法制备大体积,且性质不稳定,容易出现沉降凝絮,仍然存在声敏剂安全性等一些列问题,阻碍了声动力治疗的临床推广。However, the current nano-sound sensitizers have complicated preparation processes, cannot be prepared in large volumes, are unstable in nature, and prone to sedimentation and flocculation. There are still a series of problems such as the safety of sono-sensitizers, which hinder the clinical promotion of sonodynamic therapy.
现有技术中有报道过人血清白蛋白和载氧血红蛋白的杂化体系,但是没有加入高效且低毒性的声敏剂,无法实现深层肿瘤的高效治疗。声动力的三大因素是:超声、氧气和声敏剂。超声的深度可以高达10cm,所以当纳米颗粒渗透到肿瘤深处时,别的手段例如光,即便是二区的,也没法实现如此深层次的穿透。声敏剂在超声深穿透的情况下,可以有效地发挥作用,实现深层肿瘤的高效治疗。现有技术中也报道过人血清白蛋白包裹锰卟啉声敏剂的纳米颗粒,但是不能够高效的载氧,因此起不到高效的声动力治疗。Hybrid systems of human serum albumin and oxygen-carrying hemoglobin have been reported in the prior art, but without the addition of highly efficient and low-toxic sonosensitizers, efficient treatment of deep-seated tumors cannot be achieved. The three main factors of sonodynamics are: ultrasound, oxygen and sonosensitizer. The depth of ultrasound can be as high as 10cm, so when nanoparticles penetrate deep into the tumor, other methods such as light, even in the second zone, cannot achieve such deep penetration. Sonosensitizers can effectively function under the condition of deep ultrasound penetration to achieve efficient treatment of deep-seated tumors. Nanoparticles containing manganese porphyrin sonosensitizer coated with human serum albumin have also been reported in the prior art, but they cannot carry oxygen efficiently and therefore cannot provide efficient sonodynamic therapy.
发明内容Contents of the invention
针对现有技术中的缺陷,本发明提出了一种制备工艺简单、性质稳定、安全性高、生物利用度高、靶向性好的靶向载氧纳米声敏剂,可实现高效声动力治疗。采用二硫键“断开-重构”技术,一步超声法杂交人血清白蛋白(HSA)和载氧血红蛋白(Hb),包裹疏水的声敏剂锰卟啉(MnPP)后,制备靶向载氧纳米声敏剂,将外源氧分子和MnPP高效靶向递送到肿瘤内部,从而改善肿瘤缺氧微环境并提高声动力疗效。In view of the deficiencies in the existing technology, the present invention proposes a targeted oxygen-carrying nano-sound sensitizer with simple preparation process, stable properties, high safety, high bioavailability and good targeting property, which can achieve efficient sonodynamic therapy. . Using disulfide bond "disconnection-reconstruction" technology, one-step ultrasonic method hybridizes human serum albumin (HSA) and oxygen-carrying hemoglobin (Hb), and wraps the hydrophobic sonosensitizer manganese porphyrin (MnPP) to prepare the targeted carrier. Oxygen nano-sonosensitizer can efficiently deliver exogenous oxygen molecules and MnPP into tumors, thereby improving the tumor hypoxic microenvironment and improving sonodynamic efficacy.
本发明提供一种靶向载氧纳米声敏剂,外部为杂合蛋白体,内部包裹声敏剂锰卟啉,所述杂合蛋白体由人血清白蛋白和载氧血红蛋白组成。The invention provides a targeted oxygen-carrying nano-sound sensitizer. The outer part is a hybrid protein body and the inner part is wrapped with the sound sensitizer manganese porphyrin. The hybrid protein body is composed of human serum albumin and oxygen-carrying hemoglobin.
靶向载氧纳米声敏剂为球形结构,与具有大环共轭结构的锰卟啉声敏剂间形成较强分子间作用力,从而将锰卟啉声敏剂高效地包裹于杂合蛋白体中,提高了锰卟啉声敏剂的水溶性,从而提高其生物利用率。The targeted oxygen-carrying nano-sound sensitizer has a spherical structure and forms a strong intermolecular force with the manganese porphyrin sonosensitizer with a macrocyclic conjugated structure, thereby effectively wrapping the manganese porphyrin sonosensitizer in the hybrid protein In the body, the water solubility of the manganese porphyrin sonosensitizer is improved, thereby improving its bioavailability.
载氧血红蛋白,能够高效的载氧,可以增强声动力治疗的效果。Oxygen-carrying hemoglobin can carry oxygen efficiently and can enhance the effect of sonodynamic therapy.
人血清白蛋白具有肿瘤特异性靶向功能,通过gp60糖蛋白受体介导的肿瘤内皮细胞穿透效应以及肿瘤细胞分泌蛋白SPARC介导的增强摄取机制高效地靶向肿瘤。Human serum albumin has a tumor-specific targeting function and efficiently targets tumors through the tumor endothelial cell penetration effect mediated by the gp60 glycoprotein receptor and the enhanced uptake mechanism mediated by the tumor cell secreted protein SPARC.
进一步的,所述锰卟啉由金属锰离子与卟啉结构的大环化合物络合形成。金属锰离子与卟啉结构的大环化合物络合后,产生荧光共振能量转移效应,从而使卟啉声敏剂的荧光淬灭,降低卟啉声敏剂的光毒性。Further, the manganese porphyrin is formed by complexing a metal manganese ion with a macrocyclic compound of a porphyrin structure. After the metal manganese ion is complexed with the macrocyclic compound of the porphyrin structure, a fluorescence resonance energy transfer effect is generated, thereby quenching the fluorescence of the porphyrin sound sensitizer and reducing the phototoxicity of the porphyrin sound sensitizer.
所述的载氧纳米声敏剂,能够将外源氧分子和锰卟啉靶向递送到肿瘤内部。The oxygen-carrying nano-sound sensitizer can deliver exogenous oxygen molecules and manganese porphyrins into tumors in a targeted manner.
本发明还提供一种靶向载氧纳米声敏剂的制备方法,包括如下步骤:The invention also provides a method for preparing a targeted oxygen-carrying nano-acoustic sensitizer, which includes the following steps:
(1)锰卟啉的制备:将卟啉结构的大环化合物和金属锰盐混合,过滤沉淀,干燥;(1) Preparation of manganese porphyrin: Mix the macrocyclic compound of the porphyrin structure and the metal manganese salt, filter and precipitate, and dry;
(2)用人血清白蛋白、载氧血红蛋白和步骤(1)所得的锰卟啉制备靶向载氧纳米声敏剂。(2) Use human serum albumin, oxygen-carrying hemoglobin and the manganese porphyrin obtained in step (1) to prepare a targeted oxygen-carrying nano-sound sensitizer.
进一步的,所述步骤(1)中的所述卟啉结构的大环化合物和所述金属锰盐为等摩尔比。Further, the macrocyclic compound with the porphyrin structure and the metal manganese salt in the step (1) are in an equimolar ratio.
进一步的,所述卟啉结构的大环化合物为5,10,15,20-四(4-羧基苯基)卟啉。Further, the macrocyclic compound of the porphyrin structure is 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin.
进一步的,所述步骤(1)中的所述金属锰盐包含氯化锰、硫酸锰或醋酸锰中的至少一种。Further, the metal manganese salt in step (1) contains at least one of manganese chloride, manganese sulfate or manganese acetate.
进一步的,所述步骤(1)中包括使用惰性气体进行反应保护的步骤,所述惰性气体为氮气和氩气中的至少一种。Further, the step (1) includes the step of using an inert gas for reaction protection, and the inert gas is at least one of nitrogen and argon.
进一步的,所述步骤(1)中的所述分离步骤使用柱色谱,以硅胶作为固定相,二氯甲烷和甲醇作为流动相。Further, the separation step in step (1) uses column chromatography, using silica gel as the stationary phase and dichloromethane and methanol as the mobile phase.
进一步的,所述步骤(2)中的所述人血清白蛋白和所述载氧血红蛋白的质量比为1-5:1。这个比例会影响靶向载氧纳米声敏剂的粒径大小。Further, the mass ratio of the human serum albumin and the oxygen-carrying hemoglobin in step (2) is 1-5:1. This ratio will affect the particle size of the targeted oxygen-carrying nanosonosensitizer.
进一步的,所述步骤(2)中包括使用细胞破碎仪对所述人血清白蛋白、所述载氧血红蛋白和所述锰卟啉的混合溶液进行超声的步骤。Further, the step (2) includes the step of ultrasonicating the mixed solution of human serum albumin, oxygen-carrying hemoglobin and manganese porphyrin using a cell disruptor.
进一步的,所述超声的时间为5-20min。超声时间会影响载氧纳米声敏剂的粒径大小。Further, the ultrasonic time is 5-20 minutes. Ultrasound time will affect the particle size of oxygen-carrying nano-sound sensitizer.
进一步的,所述混合溶液的pH值为7-8。pH值直接影响载氧纳米声敏剂的粒径大小,粒径的大小直接决定了所述载氧纳米声敏剂在肿瘤部位的富集量。Further, the pH value of the mixed solution is 7-8. The pH value directly affects the particle size of the oxygen-carrying nano-sound sensitizer, and the particle size directly determines the enrichment amount of the oxygen-carrying nano-sound sensitizer in the tumor site.
本发明还提供所述的载氧纳米声敏剂在制备抗肿瘤药物中的应用。The invention also provides the application of the oxygen-carrying nano-sound sensitizer in the preparation of anti-tumor drugs.
综上,与现有技术相比,本发明达到了以下技术效果:In summary, compared with the prior art, the present invention achieves the following technical effects:
1.本发明的制备方法能够制备大体积量的靶向载氧纳米声敏剂,可以制备几升以上,现有技术的制备方法得到的纳米颗粒的体积最多几百毫升,故本发明为纳米颗粒的放大生产和临床研究提供了基础。1. The preparation method of the present invention can prepare a large volume of targeted oxygen-carrying nano-acoustic sensitizers, and can prepare more than several liters. The volume of nanoparticles obtained by the preparation method of the prior art is up to several hundred milliliters, so the present invention is a nanometer-sized sonosensitizer. Provides a basis for scale-up production and clinical research of particles.
2.本发明制备的靶向载氧纳米声敏剂能够稳定存放,一年未出现沉降、凝絮现象。2. The targeted oxygen-carrying nano-acoustic sensitizer prepared by the present invention can be stored stably without settling or flocculation for one year.
3.本发明的制备方法简便易行,在搅拌下分两步加入原材料,就可以制备出粒径均匀的靶向载氧纳米声敏剂,不需要任何高温、复杂的设备或者是苛刻的反应条件,便于操作推广。3. The preparation method of the present invention is simple and easy to implement. By adding raw materials in two steps under stirring, a targeted oxygen-carrying nano-acoustic sensitizer with uniform particle size can be prepared. It does not require any high temperature, complicated equipment or harsh reactions. conditions for easy operation and promotion.
4.本发明的金属锰离子与卟啉结构的大环化合物络合后,降低卟啉声敏剂的光毒性,提高了安全性。4. After the metal manganese ions of the present invention are complexed with the macrocyclic compound of the porphyrin structure, the phototoxicity of the porphyrin sound sensitizer is reduced and the safety is improved.
5.本发明提高了锰卟啉声敏剂的水溶性,从而提高其生物利用率。5. The present invention improves the water solubility of the manganese porphyrin sound sensitizer, thereby improving its bioavailability.
6.人血清白蛋白具有肿瘤特异性靶向功能,故本发明的载氧纳米声敏剂靶向性好。6. Human serum albumin has tumor-specific targeting function, so the oxygen-carrying nano-sound sensitizer of the present invention has good targeting properties.
附图说明Description of the drawings
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and therefore do not It should be regarded as a limitation of the scope. For those of ordinary skill in the art, other relevant drawings can also be obtained based on these drawings without exerting creative efforts.
图1为实施例4中锰卟啉声敏分子的UV-Vis吸收光谱图。Figure 1 is the UV-Vis absorption spectrum of the manganese porphyrin sound-sensitive molecule in Example 4.
图2为实施例4中靶向载氧纳米声敏剂的透射电镜图。Figure 2 is a transmission electron microscope image of the targeted oxygen-carrying nano-sound sensitizer in Example 4.
图3为实施例5中本发明的靶向载氧纳米声敏剂有效携带氧分子进入肿瘤的结果图。Figure 3 is a diagram showing the results of the targeted oxygen-carrying nanosonosensitizer of the present invention effectively carrying oxygen molecules into tumors in Example 5.
图4为实施例6中本发明的靶向载氧纳米声敏剂有效抑制肿瘤生长的结果图。Figure 4 is a diagram showing the results of effective inhibition of tumor growth by the targeted oxygen-carrying nano-sound sensitizer of the present invention in Example 6.
具体实施方式Detailed ways
为了使本技术领域的人员更好地理解本发明方案,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分的实施例,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的前提下所获得的所有其他实施例,都应当属于本发明保护的范围。In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only These are some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts should fall within the scope of protection of the present invention.
本发明的靶向载氧纳米声敏剂能够实现安全高效靶向肿瘤内部,从而改善肿瘤缺氧微环境并提高声动力疗效的原理是:The principle that the targeted oxygen-carrying nano-sonophonic agent of the present invention can achieve safe and efficient targeting inside the tumor, thereby improving the hypoxic microenvironment of the tumor and improving the sonodynamic efficacy is:
1.金属锰离子与卟啉结构的大环化合物络合后,产生荧光共振能量转移效应,从而使卟啉声敏剂的荧光淬灭,降低卟啉声敏剂的光毒性;1. After the metal manganese ion is complexed with the macrocyclic compound of the porphyrin structure, a fluorescence resonance energy transfer effect is generated, thereby quenching the fluorescence of the porphyrin sound sensitizer and reducing the phototoxicity of the porphyrin sound sensitizer;
2.卟啉分子金属配位后,超声作用生成的金属卟啉三重态(3MTTP*)与氧分子有效结合,显著地提高了金属卟啉的活性氧产率,是一类理想的声敏剂分子;2. After metal coordination of porphyrin molecules, the metalloporphyrin triplet state (3MTTP*) generated by ultrasound is effectively combined with oxygen molecules, which significantly increases the active oxygen production rate of metalloporphyrin, making it an ideal sonosensitizer. molecular;
3.靶向载氧纳米声敏剂为球形结构,与具有大环共轭结构的锰卟啉声敏剂间形成较强分子间作用力,从而将锰卟啉声敏剂高效地包裹于杂合蛋白体中,提高了锰卟啉声敏剂的水溶性,从而提高其生物利用率;3. The targeted oxygen-carrying nano-sound sensitizer has a spherical structure and forms a strong intermolecular force with the manganese porphyrin sonosensitizer having a macrocyclic conjugated structure, thereby effectively wrapping the manganese porphyrin sonosensitizer in the complex. In the protein complex, the water solubility of the manganese porphyrin sonosensitizer is improved, thereby improving its bioavailability;
4.本发明中采用了人体内的组分:人血清白蛋白和载氧血红蛋白作为杂合载体,具有很高的安全性;4. The present invention uses components in the human body: human serum albumin and oxygen-carrying hemoglobin as hybrid carriers, which are highly safe;
5.人血清白蛋白具有肿瘤特异性靶向功能:通过gp60糖蛋白受体介导的肿瘤内皮细胞穿透效应以及肿瘤细胞分泌蛋白SPARC介导的增强摄取机制高效地靶向肿瘤;5. Human serum albumin has a tumor-specific targeting function: it can efficiently target tumors through the tumor endothelial cell penetration effect mediated by the gp60 glycoprotein receptor and the enhanced uptake mechanism mediated by the tumor cell secreted protein SPARC;
6.由靶向载氧纳米声敏剂降解而释放的锰离子可用于磁共振成像。6. The manganese ions released by the degradation of targeted oxygen-carrying nanosonosensitizers can be used for magnetic resonance imaging.
本发明靶向载氧纳米声敏剂的制备方法,是采用一步超声法制备的,随着氯仿溶剂的挥发,疏水锰卟啉被包裹在杂合蛋白体中,形成球形的靶向载氧纳米声敏剂。具体步骤如下:The preparation method of the targeted oxygen-carrying nanometer sonosensitizer of the present invention is prepared by a one-step ultrasonic method. As the chloroform solvent evaporates, the hydrophobic manganese porphyrin is wrapped in the hybrid protein body to form a spherical targeted oxygen-carrying nanometer sonosensitizer. Sound sensitizer. Specific steps are as follows:
(1)锰卟啉的制备(1) Preparation of manganese porphyrin
①将等摩尔比的卟啉结构的大环化合物(如5,10,15,20-四(4-羧基苯基)卟啉,PP),和金属锰盐(氯化锰、硫酸锰或醋酸锰至少一种)混合,在惰性气体保护下,搅拌,加热回流,反应停止,冷却至室温;① Combine an equal molar ratio of a macrocyclic compound with a porphyrin structure (such as 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin, PP) and a metal manganese salt (manganese chloride, manganese sulfate or acetic acid). At least one type of manganese) is mixed, stirred under the protection of inert gas, heated to reflux, the reaction is stopped, and cooled to room temperature;
②将沉淀过滤,去离子水洗涤,真空干燥。再用柱色谱分离,低温旋转蒸发,干燥,收集样品,得到锰卟啉(MnPP)。② Filter the precipitate, wash with deionized water, and dry in vacuum. Then use column chromatography to separate, low-temperature rotary evaporation, drying, and collect samples to obtain manganese porphyrin (MnPP).
(2)靶向载氧纳米声敏剂的制备(2) Preparation of targeted oxygen-carrying nano-sound sensitizers
①称取锰卟啉,溶于氯仿中;①Weigh the manganese porphyrin and dissolve it in chloroform;
②称取载氧血红蛋白粉末,溶于超纯水中,置于摇床中反应;②Weigh the oxygen-carrying hemoglobin powder, dissolve it in ultrapure water, and place it in a shaker for reaction;
③将人血清白蛋白和载氧血红蛋白按质量比为1-5:1混合,并移取①中的锰卟啉溶液于混合液底部;③ Mix human serum albumin and oxygen-carrying hemoglobin at a mass ratio of 1-5:1, and pipet the manganese porphyrin solution in ① at the bottom of the mixed solution;
④将上述混合液置于冰水浴中,用细胞粉碎仪超声,形成悬浊液,用调pH值至7-8,再置于超声清洗器中超声,直到悬浊液变澄清,得到靶向载氧纳米声敏剂。④ Place the above mixture in an ice water bath, use a cell crusher to sonicate to form a suspension, adjust the pH value to 7-8, and then place it in an ultrasonic cleaner to sonicate until the suspension becomes clear and the target is obtained. Oxygen-carrying nano-sound sensitizer.
实施例1靶向载氧纳米声敏剂的制备方法Example 1 Preparation method of targeted oxygen-carrying nano-sound sensitizer
具体包括以下步骤:Specifically, it includes the following steps:
(1)锰卟啉的制备(1) Preparation of manganese porphyrin
①将等摩尔比的5,10,15,20-四(4-羧基苯基)卟啉(PP),购买自百灵威科技有限公司,和氯化锰混合于无水DMF溶液中,在氮气保护下,搅拌,加热回流2h,UV-Vis吸收光谱仪监控反应进行情况,待反应液由黑红色变成草绿色,且Q带峰由4个转变成2个并伴随一定程度的红移后,反应停止,冷却至室温;① Mix equal molar ratios of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (PP), purchased from Bailingwei Technology Co., Ltd., and manganese chloride in an anhydrous DMF solution, and place under nitrogen protection Next, stir and heat to reflux for 2 hours. A UV-Vis absorption spectrometer monitors the progress of the reaction. After the reaction liquid changes from black-red to grass green, and the Q-band peaks change from 4 to 2 with a certain degree of red shift, the reaction Stop and cool to room temperature;
②将沉淀过滤,去离子水洗涤,真空干燥。再用柱色谱分离,以硅胶作为固定相,体积比为3:1的二氯甲烷和甲醇作为流动相进行分离提纯。低温旋转蒸发,干燥,收集样品,得到锰卟啉(MnPP)。② Filter the precipitate, wash with deionized water, and dry in vacuum. Then use column chromatography to separate and purify, using silica gel as the stationary phase and dichloromethane and methanol with a volume ratio of 3:1 as the mobile phase. Rotate evaporate at low temperature, dry, and collect the sample to obtain manganese porphyrin (MnPP).
(2)靶向载氧纳米声敏剂的制备(2) Preparation of targeted oxygen-carrying nano-sound sensitizers
①称取锰卟啉1mg,溶于100μL氯仿中;①Weigh 1 mg of manganese porphyrin and dissolve it in 100 μL chloroform;
②称取5mg的Hb粉末,溶于1mL超纯水中,置于摇床中反应0.5h,反应条件:37℃,100rpm;②Weigh 5 mg of Hb powder, dissolve it in 1 mL of ultrapure water, and place it in a shaker for 0.5 h. Reaction conditions: 37°C, 100 rpm;
③将HSA和Hb按质量比为1:1混合均匀,并移取①中100μL的锰卟啉溶液于混合液底部;③Mix HSA and Hb evenly at a mass ratio of 1:1, and pipet 100 μL of the manganese porphyrin solution in ① at the bottom of the mixed solution;
④将上述混合液置于冰水浴中,用细胞粉碎仪超声5min,形成悬浊液,用Na2CO3溶液调pH值至7,再置于超声清洗器中超声,直到悬浊液变澄清,得到靶向载氧纳米声敏剂。④ Place the above mixed solution in an ice water bath, use a cell crusher to sonicate for 5 minutes to form a suspension, use Na 2 CO 3 solution to adjust the pH value to 7, and then place it in an ultrasonic cleaner to sonicate until the suspension becomes clear. , to obtain targeted oxygen-carrying nano-sound sensitizers.
通过粒度仪检测所得靶向载氧纳米声敏剂的zeta电位大于-30mV,粒径范围为50-200nm,电位和粒径的结果可以说明靶向载氧纳米声敏剂制备成功。但是靶向载氧纳米声敏剂的粒径范围稍大,粒径均一程度不高。这与人血清白蛋白和载氧血红蛋白的比例、溶液的pH值、超声的时间有关。The zeta potential of the targeted oxygen-carrying nano-sound sensitizer detected by a particle size analyzer is greater than -30mV, and the particle size range is 50-200 nm. The results of the potential and particle size can indicate that the targeted oxygen-carrying nano-sound sensitizer is successfully prepared. However, the particle size range of targeted oxygen-carrying nano-sound sensitizers is slightly larger, and the particle size uniformity is not high. This is related to the ratio of human serum albumin and oxygen-carrying hemoglobin, the pH value of the solution, and the time of ultrasound.
实施例2靶向载氧纳米声敏剂的制备方法Example 2 Preparation method of targeted oxygen-carrying nano-sound sensitizer
本实施例中人血清白蛋白和载氧血红蛋白的比例、体系的pH值、超声的时间与实施例1不同,pH值直接影响载氧纳米声敏剂的粒径大小,粒径的大小直接决定了所述载氧纳米声敏剂在肿瘤部位的富集量。超声时间直接影响载氧纳米声敏剂的粒径大小,人血清白蛋白和载氧血红蛋白的比例也会对粒径大小有影响。In this example, the ratio of human serum albumin and oxygen-carrying hemoglobin, the pH value of the system, and the ultrasound time are different from those in Example 1. The pH value directly affects the particle size of the oxygen-carrying nano-sound sensitizer, and the particle size directly determines The enrichment amount of the oxygen-carrying nanosonosensitizer in the tumor site was determined. The ultrasonic time directly affects the particle size of the oxygen-carrying nano-sound sensitizer, and the ratio of human serum albumin and oxygen-carrying hemoglobin also affects the particle size.
具体包括以下步骤:Specifically, it includes the following steps:
(1)锰卟啉的制备(1) Preparation of manganese porphyrin
①将等摩尔比的5,10,15,20-四(4-羧基苯基)卟啉(PP)和醋酸锰混合于无水DMF溶液中,在氩气保护下,搅拌,加热回流6h,UV-Vis吸收光谱仪监控反应进行情况,待反应液由黑红色变成草绿色,且Q带峰由4个转变成2个并伴随一定程度的红移后,反应停止,冷却至室温;① Mix equal molar ratios of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (PP) and manganese acetate in anhydrous DMF solution, stir under argon protection, and heat to reflux for 6 hours. A UV-Vis absorption spectrometer monitors the progress of the reaction. After the reaction liquid changes from black-red to grass green, and the Q-band peaks change from 4 to 2 with a certain degree of red shift, the reaction is stopped and cooled to room temperature;
②将沉淀过滤,去离子水洗涤,真空干燥。再用柱色谱分离,以硅胶作为固定相,体积比为3:1的二氯甲烷和甲醇作为流动相进行分离提纯。低温旋转蒸发,干燥,收集样品,得到锰卟啉(MnPP)。② Filter the precipitate, wash with deionized water, and dry in vacuum. Then use column chromatography to separate and purify, using silica gel as the stationary phase and dichloromethane and methanol with a volume ratio of 3:1 as the mobile phase. Rotate evaporate at low temperature, dry, and collect the sample to obtain manganese porphyrin (MnPP).
(2)靶向载氧纳米声敏剂的制备(2) Preparation of targeted oxygen-carrying nano-sound sensitizers
①称取锰卟啉5mg,溶于500μL氯仿中;①Weigh 5 mg of manganese porphyrin and dissolve it in 500 μL chloroform;
②称取10mg的Hb粉末,溶于5mL超纯水中,置于摇床中反应2h,反应条件:37℃,200rpm;②Weigh 10 mg of Hb powder, dissolve it in 5 mL of ultrapure water, and place it in a shaker to react for 2 hours. Reaction conditions: 37°C, 200 rpm;
③将HSA和Hb按质量比为5:1混合均匀,并移取①中500μL的锰卟啉溶液于混合液底部;③Mix HSA and Hb evenly at a mass ratio of 5:1, and pipet 500 μL of the manganese porphyrin solution in ① at the bottom of the mixed solution;
④将上述混合液置于冰水浴中,用细胞粉碎仪超声20min,形成悬浊液,用Na2CO3溶液调pH值至8,再置于超声清洗器中超声,直到悬浊液变澄清,得到靶向载氧纳米声敏剂。④ Place the above mixture in an ice water bath, use a cell crusher to sonicate for 20 minutes to form a suspension, adjust the pH value to 8 with Na 2 CO 3 solution, and then place it in an ultrasonic cleaner to sonicate until the suspension becomes clear. , to obtain targeted oxygen-carrying nano-sound sensitizers.
通过粒度仪检测所得靶向载氧纳米声敏剂的zeta电位大于-30mV,粒径范围为80-200nm。电位和粒径的结果可以说明靶向载氧纳米声敏剂制备成功。靶向载氧纳米声敏剂的粒径范围相比实施例1略有缩小,但是粒径范围仍有些大,粒径均一程度不高,这与人血清白蛋白和载氧血红蛋白的比例、溶液的pH值、超声的时间有关。可以通过继续优化以上条件制备粒径更均一的靶向载氧纳米声敏剂。The zeta potential of the targeted oxygen-carrying nano-sound sensitizer detected by a particle size analyzer is greater than -30mV, and the particle size range is 80-200nm. The results of potential and particle size can indicate the successful preparation of targeted oxygen-carrying nano-sound sensitizers. The particle size range of the targeted oxygen-carrying nano-sound sensitizer is slightly narrower than in Example 1, but the particle size range is still somewhat large, and the particle size uniformity is not high. This is inconsistent with the ratio of human serum albumin and oxygen-carrying hemoglobin, the solution It is related to the pH value and ultrasound time. Targeted oxygen-carrying nano-sound sensitizers with more uniform particle sizes can be prepared by continuing to optimize the above conditions.
实施例3靶向载氧纳米声敏剂的制备方法Example 3 Preparation method of targeted oxygen-carrying nano-sound sensitizer
本实施例中人血清白蛋白和载氧血红蛋白的比例、体系的pH值、超声的时间与实施例1不同,pH值直接影响载氧纳米声敏剂的粒径大小,粒径的大小直接决定了所述载氧纳米声敏剂在肿瘤部位的富集量。超声时间直接影响载氧纳米声敏剂的粒径大小,人血清白蛋白和载氧血红蛋白的比例也会对粒径大小有影响。In this example, the ratio of human serum albumin and oxygen-carrying hemoglobin, the pH value of the system, and the ultrasound time are different from those in Example 1. The pH value directly affects the particle size of the oxygen-carrying nano-sound sensitizer, and the particle size directly determines The enrichment amount of the oxygen-carrying nanosonosensitizer in the tumor site was determined. The ultrasonic time directly affects the particle size of the oxygen-carrying nano-sound sensitizer, and the ratio of human serum albumin and oxygen-carrying hemoglobin also affects the particle size.
具体包括以下步骤:Specifically, it includes the following steps:
(1)锰卟啉的制备(1) Preparation of manganese porphyrin
①将等摩尔比的5,10,15,20-四(4-羧基苯基)卟啉(PP)和醋酸锰混合于无水DMF溶液中,在氩气保护下,搅拌,加热回流6h,UV-Vis吸收光谱仪监控反应进行情况,待反应液由黑红色变成草绿色,且Q带峰由4个转变成2个并伴随一定程度的红移后,反应停止,冷却至室温;① Mix equal molar ratios of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (PP) and manganese acetate in anhydrous DMF solution, stir under argon protection, and heat to reflux for 6 hours. A UV-Vis absorption spectrometer monitors the progress of the reaction. After the reaction liquid changes from black-red to grass green, and the Q-band peaks change from 4 to 2 with a certain degree of red shift, the reaction is stopped and cooled to room temperature;
②将沉淀过滤,去离子水洗涤,真空干燥。再用柱色谱分离,以硅胶作为固定相,体积比为3:1的二氯甲烷和甲醇作为流动相进行分离提纯。低温旋转蒸发,干燥,收集样品,得到锰卟啉(MnPP)。② Filter the precipitate, wash with deionized water, and dry in vacuum. Then use column chromatography to separate and purify, using silica gel as the stationary phase and dichloromethane and methanol with a volume ratio of 3:1 as the mobile phase. Rotate evaporate at low temperature, dry, and collect the sample to obtain manganese porphyrin (MnPP).
(2)靶向载氧纳米声敏剂的制备(2) Preparation of targeted oxygen-carrying nano-sound sensitizers
①称取锰卟啉1mg,溶于200μL氯仿中;①Weigh 1 mg of manganese porphyrin and dissolve it in 200 μL chloroform;
②称取5mg的Hb粉末,溶于1mL超纯水中,置于摇床中反应0.5h,反应条件:37℃,100rpm;②Weigh 5 mg of Hb powder, dissolve it in 1 mL of ultrapure water, and place it in a shaker for 0.5 h. Reaction conditions: 37°C, 100 rpm;
③将HSA和Hb按质量比为3:1混合均匀,并移取①中200μL的锰卟啉溶液于混合液底部;③Mix HSA and Hb evenly at a mass ratio of 3:1, and pipet 200 μL of the manganese porphyrin solution in ① at the bottom of the mixed solution;
④将上述混合液置于冰水浴中,用细胞粉碎仪超声5min,形成悬浊液,用Na2CO3溶液调pH值至8,再置于超声清洗器中超声,直到悬浊液变澄清,得到靶向载氧纳米声敏剂。④ Place the above mixed solution in an ice water bath, use a cell crusher to sonicate for 5 minutes to form a suspension, adjust the pH value to 8 with Na 2 CO 3 solution, and then place it in an ultrasonic cleaner to sonicate until the suspension becomes clear. , to obtain targeted oxygen-carrying nano-sound sensitizers.
通过粒度仪检测所得靶向载氧纳米声敏剂的zeta电位大于-30mV,粒径范围为100-200nm,电位和粒径的结果可以说明靶向载氧纳米声敏剂制备成功。靶向载氧纳米声敏剂的粒径范围相比实施例2略有缩小,但是粒径范围仍有些大,粒径均一程度不高,这与人血清白蛋白和载氧血红蛋白的比例、溶液的pH值、超声的时间有关。可以通过继续优化以上条件制备粒径更均一的靶向载氧纳米声敏剂。The zeta potential of the targeted oxygen-carrying nano-sound sensitizer detected by a particle size analyzer is greater than -30mV, and the particle size range is 100-200 nm. The results of the potential and particle size can indicate that the targeted oxygen-carrying nano-sound sensitizer is successfully prepared. The particle size range of the targeted oxygen-carrying nano-sound sensitizer is slightly smaller than in Example 2, but the particle size range is still somewhat large, and the particle size uniformity is not high. This is inconsistent with the ratio of human serum albumin and oxygen-carrying hemoglobin, the solution It is related to the pH value and ultrasound time. Targeted oxygen-carrying nano-sound sensitizers with more uniform particle sizes can be prepared by continuing to optimize the above conditions.
实施例4靶向载氧纳米声敏剂的制备方法Example 4 Preparation method of targeted oxygen-carrying nano-sound sensitizer
本实施例中人血清白蛋白和载氧血红蛋白的比例、体系的pH值、超声的时间与实施例1不同,pH值直接影响载氧纳米声敏剂的粒径大小,粒径的大小直接决定了所述载氧纳米声敏剂在肿瘤部位的富集量。超声时间直接影响载氧纳米声敏剂的粒径大小,人血清白蛋白和载氧血红蛋白的比例也会对粒径大小有影响。In this example, the ratio of human serum albumin and oxygen-carrying hemoglobin, the pH value of the system, and the ultrasound time are different from those in Example 1. The pH value directly affects the particle size of the oxygen-carrying nano-sound sensitizer, and the particle size directly determines The enrichment amount of the oxygen-carrying nanosonosensitizer in the tumor site was determined. The ultrasonic time directly affects the particle size of the oxygen-carrying nano-sound sensitizer, and the ratio of human serum albumin and oxygen-carrying hemoglobin also affects the particle size.
具体包括以下步骤:Specifically, it includes the following steps:
(1)锰卟啉的制备(1) Preparation of manganese porphyrin
①将等摩尔比的5,10,15,20-四(4-羧基苯基)卟啉(PP)和硫酸锰混合于无水DMF溶液中,在氩气保护下,搅拌,加热回流4h,UV-Vis吸收光谱仪监控反应进行情况,待反应液由黑红色变成草绿色,且Q带峰由4个转变成2个并伴随一定程度的红移后,反应停止,冷却至室温;① Mix equal molar ratios of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (PP) and manganese sulfate in anhydrous DMF solution, stir under argon protection, and heat to reflux for 4 hours. A UV-Vis absorption spectrometer monitors the progress of the reaction. After the reaction liquid changes from black-red to grass green, and the Q-band peaks change from 4 to 2 with a certain degree of red shift, the reaction is stopped and cooled to room temperature;
②将沉淀过滤,去离子水洗涤,真空干燥。再用柱色谱分离,以硅胶作为固定相,体积比为3:1的二氯甲烷和甲醇作为流动相进行分离提纯。低温旋转蒸发,干燥,收集样品,得到锰卟啉(MnPP)。② Filter the precipitate, wash with deionized water, and dry in vacuum. Then use column chromatography to separate and purify, using silica gel as the stationary phase and dichloromethane and methanol with a volume ratio of 3:1 as the mobile phase. Rotate evaporate at low temperature, dry, and collect the sample to obtain manganese porphyrin (MnPP).
(2)靶向载氧纳米声敏剂的制备(2) Preparation of targeted oxygen-carrying nano-sound sensitizers
①称取锰卟啉3mg,溶于300μL氯仿中;①Weigh 3 mg of manganese porphyrin and dissolve it in 300 μL chloroform;
②称取7.5mg的Hb粉末,溶于3mL超纯水中,置于摇床中反应1.5h,反应条件:37℃,150rpm;②Weigh 7.5 mg of Hb powder, dissolve it in 3 mL of ultrapure water, and place it in a shaker to react for 1.5 hours. Reaction conditions: 37°C, 150 rpm;
③将HSA和Hb按质量比为3:1混合均匀,并移取①中300μL的锰卟啉溶液于混合液底部;③Mix HSA and Hb evenly at a mass ratio of 3:1, and pipet 300 μL of the manganese porphyrin solution in ① to the bottom of the mixed solution;
④将上述混合液置于冰水浴中,用细胞粉碎仪超声12.5min,形成悬浊液,用Na2CO3溶液调pH值至7.5,再置于超声清洗器中超声,直到悬浊液变澄清,得到靶向载氧纳米声敏剂。④ Place the above mixed solution in an ice water bath, use a cell crusher to sonicate for 12.5 minutes to form a suspension, adjust the pH value to 7.5 with Na 2 CO 3 solution, and then place it in an ultrasonic cleaner to sonicate until the suspension becomes After clarification, the targeted oxygen-carrying nano-sound sensitizer was obtained.
通过粒度仪检测所得靶向载氧纳米声敏剂的zeta电位大于-30mV,粒径范围为100-150nm,电位和粒径的结果可以说明靶向载氧纳米声敏剂制备成功。这样的载氧纳米声敏剂最后在体内,可以更加高效地富集到肿瘤部位。The zeta potential of the targeted oxygen-carrying nano-sound sensitizer detected by a particle size analyzer is greater than -30mV, and the particle size range is 100-150 nm. The results of the potential and particle size can indicate that the targeted oxygen-carrying nano-sound sensitizer is successfully prepared. Such oxygen-carrying nano-sound sensitizers can be more efficiently concentrated into tumor sites in the body.
粒径大小意味着负载声敏剂的量不同,导致载氧量存在差异,但是粒径的大小直接决定了纳米声敏剂在肿瘤部位的富集量,一般来说,粒径为100-150nm时,该靶向载氧纳米声敏剂更容易富集到肿瘤部位。The particle size means that the amount of loaded sonosensitizer is different, resulting in differences in oxygen carrying capacity. However, the size of the particle size directly determines the enrichment of the nano sonosensitizer in the tumor site. Generally speaking, the particle size is 100-150nm. , the targeted oxygen-carrying nano-sound sensitizer is more likely to be enriched in the tumor site.
在实施例1-4中,制备得到的载氧纳米声敏剂的粒径大小有不同,影响粒径大小的因素有:人血清白蛋白和载氧血红蛋白的比例、体系的pH值、超声的时间。本发明摸索了各种制备条件,图1为本实施例中锰卟啉声敏分子的UV-Vis吸收光谱图,图中可以看出制备得到的锰卟啉的Q带峰由4个转变成2个并伴随一定程度的红移(见图1的虚线框),说明本发明的制备方法可以有效地制备出锰卟啉声敏分子。图2为本实施例中靶向载氧纳米声敏剂的透射电镜图,图中可以看出制备得到的靶向载氧纳米声敏剂形状规则,粒径为100-150nm,说明本发明的制备方法可以有效的制备出靶向载氧纳米声敏剂。In Examples 1-4, the prepared oxygen-carrying nano-sound sensitizers have different particle sizes. The factors that affect the particle size include: the ratio of human serum albumin and oxygen-carrying hemoglobin, the pH value of the system, the ultrasonic time. The present invention explored various preparation conditions. Figure 1 is the UV-Vis absorption spectrum of the manganese porphyrin sound-sensitive molecule in this embodiment. It can be seen from the figure that the Q-band peaks of the prepared manganese porphyrin changed from 4 to 2 and accompanied by a certain degree of red shift (see the dotted box in Figure 1), indicating that the preparation method of the present invention can effectively prepare manganese porphyrin sound-sensitive molecules. Figure 2 is a transmission electron microscope image of the targeted oxygen-carrying nano-sound sensitizer in this embodiment. It can be seen from the figure that the prepared targeted oxygen-carrying nano-sound sensitizer has a regular shape and a particle size of 100-150 nm, illustrating that the present invention The preparation method can effectively prepare targeted oxygen-carrying nano-sound sensitizers.
本实施例制备得到的靶向载氧纳米声敏剂颗粒最均一、载氧量最高、可以更好地靶向蓄积到肿瘤部位。故本实施例的制备条件和比例是最优选的。动物实验结果也可以说明,具体见实施例5和实施例6。The targeted oxygen-carrying nano-sound sensitizer particles prepared in this embodiment are the most uniform, have the highest oxygen-carrying capacity, and can be better targeted and accumulated at tumor sites. Therefore, the preparation conditions and proportions of this embodiment are the most preferred. The results of animal experiments can also be explained, see Example 5 and Example 6 for details.
实施例5本发明的靶向载氧纳米声敏剂有效携带氧分子进入肿瘤Example 5 The targeted oxygen-carrying nano-sound sensitizer of the present invention can effectively carry oxygen molecules into tumors
用实施例4制备得到的靶向载氧纳米声敏剂尾静脉注射到荷瘤小鼠(可直接购买得到)中,注射前和注射3h后,采用小动物光声仪测定,结果如图3所示,对照组为只加磷酸缓冲液的对照组,结果显示本发明的靶向载氧纳米声敏剂比对照组有显著的光声信号(如白色箭头所指区域),故本发明的靶向载氧纳米声敏剂可以有效地改善肿瘤缺氧微环境。The targeted oxygen-carrying nano-sound sensitizer prepared in Example 4 was injected into the tail vein of tumor-bearing mice (which can be purchased directly). Before the injection and 3 hours after the injection, the small animal photoacoustic instrument was used to measure the results. The results are shown in Figure 3 As shown, the control group is a control group that only adds phosphate buffer. The results show that the targeted oxygen-carrying nano-sound sensitizer of the present invention has a significant photoacoustic signal (such as the area pointed by the white arrow) compared with the control group. Therefore, the Targeted oxygen-carrying nanosonosensitizers can effectively improve the tumor hypoxic microenvironment.
实施例6本发明的靶向载氧纳米声敏剂能够有效地抑制肿瘤生长Example 6 The targeted oxygen-carrying nano-sound sensitizer of the present invention can effectively inhibit tumor growth
用实施例4制备得到的靶向载氧纳米声敏剂尾静脉注射入荷瘤小鼠(可直接购买得到),3小时后,开始超声治疗5分钟,治疗后20天,杀死小鼠,取出肿瘤测量肿瘤的体积,结果如图4所示,PBS为只加磷酸缓冲液的对照组,US代表超声,结果显示本发明的靶向载氧纳米声敏剂在超声的作用下肿瘤体积比对照组明显减小,故本发明的靶向载氧纳米声敏剂可以有效地抑制肿瘤生长。The targeted oxygen-carrying nano-sound sensitizer prepared in Example 4 was injected into the tail vein of tumor-bearing mice (which can be purchased directly). After 3 hours, ultrasonic treatment was started for 5 minutes. 20 days after treatment, the mice were killed and taken out. The volume of the tumor was measured. The results are shown in Figure 4. PBS is the control group with only phosphate buffer added, and US represents ultrasound. The results show that the targeted oxygen-carrying nano-sound sensitizer of the present invention has a better tumor volume than the control under the action of ultrasound. The group is significantly reduced, so the targeted oxygen-carrying nano-sound sensitizer of the present invention can effectively inhibit tumor growth.
综上所述,本发明采用一步超声法杂交人血清白蛋白(HSA)和载氧血红蛋白(Hb),包裹疏水的声敏剂锰卟啉(MnPP),制备靶向载氧纳米声敏剂。制备工艺简单,制备得到的靶向载氧纳米声敏剂性质稳定、生物利用度高、靶向性好、能够将外源氧分子和MnPP高效靶向递送到肿瘤内部,改善肿瘤缺氧微环境,提高声动力疗效。In summary, the present invention uses a one-step ultrasonic method to hybridize human serum albumin (HSA) and oxygen-carrying hemoglobin (Hb), and wrap the hydrophobic sonosensitizer manganese porphyrin (MnPP) to prepare a targeted oxygen-carrying nano-sound sensitizer. The preparation process is simple, and the prepared targeted oxygen-carrying nano-sonophore has stable properties, high bioavailability, and good targeting properties. It can efficiently deliver exogenous oxygen molecules and MnPP to the inside of the tumor and improve the tumor hypoxic microenvironment. , improve the sonodynamic efficacy.
1.本发明的制备方法能够制备大体积量的靶向载氧纳米声敏剂,可以制备几升以上,现有技术的制备方法得到的纳米颗粒的体积最多几百毫升,故本发明为纳米颗粒的放大生产和临床研究提供了基础。1. The preparation method of the present invention can prepare a large volume of targeted oxygen-carrying nano-acoustic sensitizers, and can prepare more than several liters. The volume of nanoparticles obtained by the preparation method of the prior art is up to several hundred milliliters, so the present invention is a nanometer-sized sonosensitizer. Provides a basis for scale-up production and clinical research of particles.
2.本发明制备的靶向载氧纳米声敏剂能够稳定存放,一年未出现沉降、凝絮现象。2. The targeted oxygen-carrying nano-acoustic sensitizer prepared by the present invention can be stored stably without settling or flocculation for one year.
3.本发明的制备方法简便易行,在搅拌下分两步加入原材料,就可以制备出粒径均匀的靶向载氧纳米声敏剂,不需要任何高温、复杂的设备或者是苛刻的反应条件,便于操作推广。3. The preparation method of the present invention is simple and easy to implement. By adding raw materials in two steps under stirring, a targeted oxygen-carrying nano-acoustic sensitizer with uniform particle size can be prepared. It does not require any high temperature, complicated equipment or harsh reactions. conditions for easy operation and promotion.
4.本发明的金属锰离子与卟啉结构的大环化合物络合后,降低卟啉声敏剂的光毒性,提高了安全性。4. After the metal manganese ions of the present invention are complexed with the macrocyclic compound of the porphyrin structure, the phototoxicity of the porphyrin sound sensitizer is reduced and the safety is improved.
5.本发明提高了锰卟啉声敏剂的水溶性,从而提高其生物利用率。5. The present invention improves the water solubility of the manganese porphyrin sound sensitizer, thereby improving its bioavailability.
6.人血清白蛋白具有肿瘤特异性靶向功能,故本发明的载氧纳米声敏剂靶向性好。6. Human serum albumin has tumor-specific targeting function, so the oxygen-carrying nano-sound sensitizer of the present invention has good targeting properties.
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.
Claims (5)
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