CN111529704A

Movatterモバイル変換

Info

Publication number: CN111529704A
Application number: CN202010197178.5A
Authority: CN
Inventors: 黄曦; 廖玉辉; 李斌
Original assignee: Fifth Affiliated Hospital of Sun Yat Sen University
Current assignee: Fifth Affiliated Hospital of Sun Yat Sen University
Priority date: 2020-03-19
Filing date: 2020-03-19
Publication date: 2020-08-14
Anticipated expiration: 2040-03-19
Also published as: CN111529704B

Abstract

Translated fromChinese

本发明属于靶向成像和治疗技术领域，公开了一种聚集发光光敏剂/抗菌药物多功能纳米胶束及其制备方法和应用，该纳米胶束为核‑壳结构，其核由聚集发光光敏剂TTD和抗结核药物组成，壳为单层脂质膜，多功能纳米胶束的直径为100‑120nm；抗结核药物为利福平。纳米胶束内部的聚集发光光敏剂TTD能够产生大量ROS氧自由基用于诱导结核菌的光动力杀伤，胶束内部缓慢释放的抗结核药物利福平又能进一步抑制结核菌的生长，从而发挥光动力、药物协同治疗功能。该多功能纳米胶束不仅可作为造影剂用于结核病灶定位，还可作为治疗剂用于结核菌的协同杀伤。

The invention belongs to the technical field of targeted imaging and treatment, and discloses an aggregated luminescence photosensitizer/antibacterial drug multifunctional nano-micelle and a preparation method and application thereof. It is composed of TTD and an anti-tuberculosis drug, the shell is a monolayer lipid film, and the diameter of the multifunctional nanomicelle is 100-120 nm; the anti-tuberculosis drug is rifampicin. The aggregated luminescent photosensitizer TTD inside the nanomicelles can generate a large number of ROS oxygen radicals to induce the photodynamic killing of Mycobacterium tuberculosis, and the anti-tuberculosis drug rifampicin slowly released inside the micelles can further inhibit the growth of Mycobacterium tuberculosis. Photodynamic, drug synergistic treatment function. The multifunctional nanomicelle can be used not only as a contrast agent for localization of tuberculosis lesions, but also as a therapeutic agent for the synergistic killing of tuberculosis bacteria.

Description

Translated fromChinese

一种聚集发光光敏剂/抗菌药物多功能纳米胶束及其制备方法和应用A kind of aggregated luminescent photosensitizer/antibacterial drug multifunctional nanomicelle and preparation method thereoflaw and application

技术领域technical field

本发明涉及靶向成像和治疗技术领域，更具体地，涉及一种聚集发光光敏剂/抗菌药物多功能纳米胶束及其制备方法和应用。The invention relates to the technical field of targeted imaging and treatment, and more particularly, to an aggregated luminescent photosensitizer/antibacterial drug multifunctional nanomicelle and a preparation method and application thereof.

背景技术Background technique

结核病(Tuberculosis，TB)是一种由结核分枝杆菌(Mycobacteriumtuberculosis，M.tb)感染引发的慢性传染性疾病，严重威胁人类健康。据《2018年全球结核报告》统计，2017年全球新发结核病例约1000万，年死亡人数达160余万。更可怕的是，现在全世界有超过23％的人口(约17亿人)为结核潜伏感染者，其中有约10％将在其一生中患上结核病。抗生素治疗作为现在市面上唯一的结核病治疗方法，有给药时间长，毒副作用显著，患者依从性差等缺点和不足。并且，多耐药结核菌(MDR-M.tb)和泛耐药结核菌(XDR-M.tb)的大量出现显著降低了抗生素的疗效。因此，开发新型结核病治疗方法，对于降低结核病的死亡率，提高结核病患者的生活质量具有重要意义。Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (M.tb) infection, which seriously threatens human health. According to the "2018 Global Tuberculosis Report", there were about 10 million new cases of tuberculosis in the world in 2017, and the annual death toll reached more than 1.6 million. What is even more frightening is that more than 23% of the world's population (about 1.7 billion people) are latent tuberculosis infection, and about 10% of them will develop tuberculosis in their lifetime. Antibiotic therapy, as the only treatment method for tuberculosis on the market today, has shortcomings and deficiencies such as long administration time, significant toxic and side effects, and poor patient compliance. Moreover, the emergence of multidrug-resistant tuberculosis (MDR-M.tb) and pan-drug-resistant tuberculosis (XDR-M.tb) significantly reduced the efficacy of antibiotics. Therefore, the development of new treatment methods for tuberculosis is of great significance for reducing the mortality of tuberculosis and improving the quality of life of tuberculosis patients.

结核结节作为结核病的特征性病灶，由紧密排列的免疫细胞聚集组成，中央包绕大量死亡或未死亡的结核杆菌。起初，人们认为结核结节能够将结核菌隔绝在内部而抑制其生长繁殖。但是，有证据表明，初期结节能够促进结核菌的扩散；结核菌的致密结构限制了抗生素的进入；结节内部处于静息状态的结核菌本身对抗生素不敏感。这都致使结核病成为一种慢性且迁延不愈的传染性疾病。因此，以纳米材料介导的光动力学疗法协同化疗将能够解决抗单纯生素治疗的固有缺陷，可能成为一种结核病治疗新策略。Tuberculosis nodules, the characteristic foci of tuberculosis, consist of clusters of tightly packed immune cells surrounded by a large number of dead or undead Mycobacterium tuberculosis. At first, it was thought that tuberculosis nodules could keep TB bacteria inside and inhibit their growth and reproduction. However, there is evidence that early nodules can promote the spread of TB; the dense structure of TB restricts the entry of antibiotics; and the quiescent TB bacteria inside the nodules are themselves insensitive to antibiotics. All this makes TB a chronic and persistent infectious disease. Therefore, synergistic chemotherapy with nanomaterial-mediated photodynamic therapy will be able to solve the inherent defects of antibiotics alone, and may become a new strategy for the treatment of tuberculosis.

发明内容SUMMARY OF THE INVENTION

本发明针对现有技术存在的上述缺陷，首先提供一种用于结核病靶向成像和多模态治疗的多功能纳米胶束。Aiming at the above-mentioned defects in the prior art, the present invention first provides a multifunctional nanomicelle for tuberculosis targeted imaging and multimodal treatment.

本发明的第二个目的是提供上述多功能纳米胶束的制备方法。The second object of the present invention is to provide a method for preparing the above-mentioned multifunctional nanomicelles.

本发明的第三个目的是提供上述多功能纳米胶束的应用。The third object of the present invention is to provide the application of the above-mentioned multifunctional nanomicelles.

本发明的目的通过以下技术方案予以实现：The object of the present invention is achieved through the following technical solutions:

一种用于结核病靶向成像和多模态治疗的多功能纳米胶束，其特征在于，所述多功能纳米胶束为核-壳结构，其核由聚集发光光敏剂TTD和抗结核药物组成，所述壳为单层脂质膜，该多功能纳米胶束的直径为100-120nm；所述抗结核药物为利福平。A multifunctional nanomicelle for tuberculosis targeted imaging and multimodal treatment, characterized in that the multifunctional nanomicelle has a core-shell structure, the core of which is composed of an aggregated luminescent photosensitizer TTD and an anti-tuberculosis drug , the shell is a monolayer lipid film, the diameter of the multifunctional nano micelle is 100-120 nm; the anti-tuberculosis drug is rifampicin.

本发明以聚集发光光敏分子TTD与抗结核药物RIF为共载体，形成纳米胶束的核心，以独特的设计方案，将脂质体膜，用于包埋TTD与RIF，并设计合成TTD/RIF@lipid-PEG纳米胶束，获得具有聚集发光成像及可见光激发同时发挥结核菌光动力、药物功能的多功能纳米胶束。该TTD/RIF@lipid-PEG纳米胶束具有100-120nm的直径，其等同于存在于人和动物机体中的一些蛋白和生物有机化合物的尺寸，由此便于造影剂在关注区域的输送和吸收。In the invention, the aggregated luminescent photosensitive molecule TTD and anti-tuberculosis drug RIF are used as co-carriers to form the core of nano micelles, and the liposome membrane is used for embedding TTD and RIF with a unique design scheme, and TTD/RIF is designed and synthesized @lipid-PEG nanomicelles have obtained multifunctional nanomicelles with aggregated luminescence imaging and visible light excitation, which simultaneously exert photodynamic and drug functions of tuberculosis. The TTD/RIF@lipid-PEG nanomicelles have a diameter of 100-120 nm, which is equivalent to the size of some proteins and bioorganic compounds present in human and animal bodies, thereby facilitating the delivery and absorption of contrast agents in the region of interest .

本发明的多功能纳米胶束中，聚集发光光敏剂TTD为一种结构对称的聚集发光材料，具有聚集诱导发光的特性；相比出传统的拥有聚集诱导淬灭现象的荧光分子(如芘)，聚集发光剂能够在聚集状态下发射强烈荧光，因此更适用于生物学领域的荧光成像；并且，本发明中TTD通过疏水相互作用被包裹于纳米胶束核心，处于更加强烈的聚集状态，因此会发射更加强烈的荧光。本发明中的TTD分子还具有光敏剂的特性，可被可见光激发，并与分子氧作用，产生氧自由基(ROS)用于结核菌的光动力杀伤。本发明中的抗菌药物RIF为抗结核一线药物，能够抑制细菌细胞壁生成，抑制结核菌生长繁殖。In the multifunctional nanomicelle of the present invention, the aggregated luminescence photosensitizer TTD is a kind of aggregated luminescent material with symmetrical structure, and has the property of aggregation-induced luminescence; , the aggregated luminescent agent can emit strong fluorescence in the aggregated state, so it is more suitable for fluorescence imaging in the field of biology; and in the present invention, TTD is wrapped in the core of the nanomicelle through hydrophobic interaction, and is in a more intense aggregation state, so emits more intense fluorescence. The TTD molecule in the present invention also has the characteristics of a photosensitizer, which can be excited by visible light and interact with molecular oxygen to generate oxygen free radicals (ROS) for photodynamic killing of Mycobacterium tuberculosis. The antibacterial drug RIF in the present invention is a first-line drug against tuberculosis, which can inhibit the formation of bacterial cell walls and inhibit the growth and reproduction of tuberculosis bacteria.

本发明还提供所述的多功能纳米胶束的制备方法，包括以下步骤：The present invention also provides a method for preparing the multifunctional nanomicelle, comprising the following steps:

(1)将利福平溶解在THF溶剂中，并加入聚集发光光敏剂TTD和双亲性胶束分子聚乙二醇2000-二硬脂酰基磷脂酰乙醇胺形成THF混合液；其中，聚集发光光敏剂TTD、利福平和双亲性胶束分子聚乙二醇2000-二硬脂酰基磷脂酰乙醇胺的质量比为1-2：12-30：2-4。(1) Dissolving rifampicin in THF solvent, and adding aggregated luminescent photosensitizer TTD and amphiphilic micellar molecule polyethylene glycol 2000-distearoylphosphatidylethanolamine to form a THF mixed solution; wherein, aggregated luminescent photosensitizer The mass ratio of TTD, rifampicin and the amphiphilic micellar molecule polyethylene glycol 2000-distearoylphosphatidylethanolamine was 1-2:12-30:2-4.

(2)将THF混合液注入超纯水中，经超声、避光磁力搅拌后，蒸干THF溶剂；(2) The THF mixed solution is injected into ultrapure water, and after ultrasonic and light-proof magnetic stirring, the THF solvent is evaporated to dryness;

(3)经多次过滤，洗去未结合的利福平和双亲性胶束分子聚乙二醇2000-二硬脂酰基磷脂酰乙醇胺，即得TTD/RIF@lipid-PEG多功能纳米胶束。(3) After multiple filtrations, the unbound rifampicin and the amphiphilic micellar molecule polyethylene glycol 2000-distearoylphosphatidylethanolamine were washed away to obtain TTD/RIF@lipid-PEG multifunctional nanomicelles.

本发明的多功能纳米胶束合成原料中，聚乙二醇2000-二硬脂酰基磷脂酰乙醇胺(DSPE-PEG2000)具有双亲性特性，聚集发光光敏分子TTD具有疏水性，而抗结核药物RIF具有水微溶性。因此，在探头超声的作用下，DSPE-PEG₂₀₀₀的疏水端DSPE会自我聚集，并包裹疏水性物质TTD和微溶于水的RIF形成纳米胶束核心，DSPE-PEG₂₀₀₀的疏水端PEG₂₀₀₀会分布与纳米胶束表面，维持此结构的稳定性。Among the multifunctional nanomicelle synthesis raw materials of the present invention, polyethylene glycol 2000-distearoyl phosphatidylethanolamine (DSPE-PEG2000) has amphiphilic properties, the aggregated light-emitting photosensitizer TTD has hydrophobicity, and the anti-tuberculosis drug RIF has Water slightly soluble. Therefore, under the action of probe ultrasound, the hydrophobic end DSPE of DSPE-PEG₂₀₀₀ will self-aggregate and encapsulate the hydrophobic substance TTD and slightly water-soluble RIF to form a nanomicelle core, and the hydrophobic end PEG_{2000 of DSPE-PEG 2000}_will distribution with the nanomicelle surface, maintaining the stability of this structure.

上述构建的TTD/RIF@lipid-PEG多功能纳米胶束具有较高的生物相容性与光照稳定性等特点。为提高纳米胶束的体内循环时间，降低网状内皮系统(Reticuloendothelialsystem)的吞噬作用，本发明采用FDA批准的、已用于临床的DSPE-PEG₂₀₀₀(聚乙二醇-二硬脂酰基磷脂酰乙醇胺)脂质体膜，其可有效提高药物体循环时间。The above constructed TTD/RIF@lipid-PEG multifunctional nanomicelles have the characteristics of high biocompatibility and light stability. In order to improve the in vivo circulation time of nano micelles and reduce the phagocytosis of the reticuloendothelial system, the present invention adopts FDA-approved and clinically used DSPE-PEG₂₀₀₀ (polyethylene glycol-distearoyl phosphatidyl group). ethanolamine) liposome membrane, which can effectively improve the drug system circulation time.

优选的，步骤(1)聚集发光光敏剂TTD、利福平和双亲性胶束分子聚乙二醇2000-二硬脂酰基磷脂酰乙醇胺的质量比为1：20：2。Preferably, the mass ratio of the aggregated luminescent photosensitizer TTD, rifampicin and the amphiphilic micelle molecule polyethylene glycol 2000-distearoylphosphatidylethanolamine in step (1) is 1:20:2.

优选的，步骤(2)超声的条件为60-80W，20-25kHz，时间为1-5min。Preferably, the ultrasonic conditions in step (2) are 60-80W, 20-25kHz, and the time is 1-5min.

优选的，步骤(3)所述多次过滤是先经过0.22μm孔径的针头过滤器过滤，后经过10KDa的超滤进行超滤。Preferably, the multiple filtrations in step (3) are firstly filtered through a needle filter with a pore size of 0.22 μm, and then subjected to ultrafiltration with a 10KDa ultrafiltration.

本发明还提供一种用于治疗结核病的制剂，所述制剂包括上述的多功能纳米胶束。该多功能纳米胶束具备对结核病灶结核结节的荧光成像功能，实现结核病灶的靶向，并且兼有对结核菌的光动力杀伤与药物杀伤协同治疗功能。The present invention also provides a preparation for treating tuberculosis, the preparation comprising the above-mentioned multifunctional nanomicelles. The multifunctional nanomicelle has the function of fluorescence imaging of tuberculosis nodules in tuberculosis lesions, realizes the targeting of tuberculosis lesions, and has both photodynamic killing and drug killing synergistic treatment functions for tuberculosis bacteria.

本发明还提供多功能纳米胶束在制备用于结核病灶成像的造影剂的应用。The invention also provides the application of the multifunctional nano micelles in the preparation of contrast agents for imaging tuberculosis lesions.

或者，本发明还提供多功能纳米胶束在制备用于结核菌光动力、药物协同杀伤剂中的应用。所述的TTD/RIF@lipid-PEG多功能纳米胶束，本发明TTD/RIF@lipid-PEG多功能纳米胶束可用于结核结节的荧光成像；而且由于灵敏度的提高，可以降低造影剂用量，进一步减轻毒副作用。在可见光照射下，内部包埋的TTD聚集发光光敏分子，能够有效的将光能转化化学能，产生大量ROS氧自由基，进而用于杀伤结核杆菌；同时，内部包含的抗结核药物RIF会缓慢释放，抑制结核菌的生长。Alternatively, the present invention also provides the application of the multifunctional nanomicelle in the preparation of a photodynamic and drug synergistic killing agent for Mycobacterium tuberculosis. The TTD/RIF@lipid-PEG multifunctional nanomicelle, the TTD/RIF@lipid-PEG multifunctional nanomicelle of the present invention can be used for fluorescence imaging of tuberculosis nodules; and due to the improvement of sensitivity, the dosage of contrast agent can be reduced , to further reduce the toxic and side effects. Under visible light irradiation, the embedded TTD aggregates light-emitting photosensitive molecules, which can effectively convert light energy into chemical energy and generate a large number of ROS oxygen free radicals, which are then used to kill Mycobacterium tuberculosis; at the same time, the anti-tuberculosis drug RIF contained inside will slowly release, inhibits the growth of tuberculosis bacteria.

本发明所构建的TTD/RIF@lipid-PEG多功能纳米胶束可通过任何已知的递送方法方式给药：全身递送(静脉注射)，动脉内，胃肠外，肺腔内或局部给药的区域递送形式。如通过静脉注射方式给药，能够实现病灶部位的靶向成像及成像介导的光动力、药物治疗。The TTD/RIF@lipid-PEG multifunctional nanomicelles constructed in the present invention can be administered by any known delivery method: systemic delivery (intravenous injection), intraarterial, parenteral, intrapulmonary or local administration regional delivery form. If administered by intravenous injection, targeted imaging of the lesion site and imaging-mediated photodynamic and drug therapy can be achieved.

与现有技术相比，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:

本发明的多功能纳米胶束中，聚集发光光敏剂TTD为一种结构对称的聚集发光材料，具有聚集诱导发光的特性；相比出传统的拥有聚集诱导淬灭现象的荧光分子(如芘)，聚集发光剂能够在聚集状态下发射强烈荧光，因此更适用于生物学领域的荧光成像；并且，本发明中TTD通过疏水相互作用被包裹于纳米胶束核心，处于更加强烈的聚集状态，因此会发射更加强烈的荧光。本发明中的TTD分子还具有光敏剂的特性，可被可见光激发，并与分子氧作用，产生氧自由基(ROS)用于结核菌的光动力杀伤。制备得到的多功能纳米胶束不仅可用于结核病灶的靶向成像，还可作为结核杆菌的光动力、药物协同治疗应用。发明还可解决结核病传统抗生素治疗方案中疗效差，给药时间长，毒副作用显著，患者依从性差等缺点和不足，成为一种结核病灶靶向及结核病治疗新策略。In the multifunctional nanomicelle of the present invention, the aggregated luminescence photosensitizer TTD is a kind of aggregated luminescent material with symmetrical structure, and has the property of aggregation-induced luminescence; , the aggregated luminescent agent can emit strong fluorescence in the aggregated state, so it is more suitable for fluorescence imaging in the field of biology; and in the present invention, TTD is wrapped in the core of the nanomicelle through hydrophobic interaction, and is in a more intense aggregation state, so emits more intense fluorescence. The TTD molecule in the present invention also has the characteristics of a photosensitizer, which can be excited by visible light and interact with molecular oxygen to generate oxygen free radicals (ROS) for photodynamic killing of Mycobacterium tuberculosis. The prepared multifunctional nanomicelles can not only be used for targeted imaging of tuberculosis lesions, but also can be used for photodynamic and drug synergistic treatment of Mycobacterium tuberculosis. The invention can also solve the shortcomings and deficiencies of the traditional antibiotic treatment scheme for tuberculosis, such as poor curative effect, long administration time, significant toxic and side effects, and poor patient compliance, and becomes a new strategy for tuberculosis focus targeting and tuberculosis treatment.

附图说明Description of drawings

图1为基于TTD/RIF@lipid-PEG多功能纳米胶束的结核病靶向成像和治疗方法原理图；Figure 1 is a schematic diagram of the TTD/RIF@lipid-PEG multifunctional nanomicelle-based targeted imaging and treatment method for tuberculosis;

图2为聚集发光光敏剂TTD的化学合成路线；Fig. 2 is the chemical synthesis route of aggregated luminescent photosensitizer TTD;

图3为TTD/RIF@lipid-PEG多功能纳米胶束的透射电镜图像(图3A)及水合粒径和zeta电位图(图3B)；Fig. 3 is a transmission electron microscope image of TTD/RIF@lipid-PEG multifunctional nanomicelles (Fig. 3A), as well as hydrated particle size and zeta potential (Fig. 3B);

图4为TTD/RIF@lipid-PEG多功能纳米胶束的紫外可见吸收光谱曲线图及发射光谱曲线图；Fig. 4 shows the UV-Vis absorption and emission spectra of TTD/RIF@lipid-PEG multifunctional nanomicelles;

图5为TTD/RIF@lipid-PEG多功能纳米胶束的体外光照ROS氧自由基产生效率图(ABDA为ROS产生指示剂)；Figure 5 is a graph showing the generation efficiency of TTD/RIF@lipid-PEG multifunctional nanomicelles in vitro for ROS oxygen radical generation (ABDA is an indicator of ROS generation);

图6为TTD/RIF@lipid-PEG多功能纳米胶束对海分枝杆菌感染斑马鱼胚胎局部结核结节模型中结节的靶向成像图；Figure 6 shows the targeted imaging of TTD/RIF@lipid-PEG multifunctional nanomicelles on the nodules in the local tuberculosis nodule model of zebrafish embryos infected with Mycobacterium marinum;

图7为TTD/RIF@lipid-PEG多功能纳米胶束对海分枝杆菌感染小鼠尾部结节模型中结节的靶向成像图；Figure 7 shows the targeted imaging of TTD/RIF@lipid-PEG multifunctional nanomicelles on nodules in a mouse tail nodule model infected with Mycobacterium marinum;

图8为TTD/RIF@lipid-PEG多功能纳米胶束对结核菌的杀伤效果CFU涂板(左图)及CFU计数统计图(右图)。Figure 8 shows the killing effect of TTD/RIF@lipid-PEG multifunctional nanomicelles on Mycobacterium tuberculosis CFU coating (left image) and CFU count statistics (right image).

具体实施方式Detailed ways

为了对本发明的技术特征、目的和效果有更加清楚的理解，现对照附图详细说明本发明的具体实施方式。显然，所描述的实施例仅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。In order to have a clearer understanding of the technical features, objects and effects of the present invention, the specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings. Obviously, the described embodiments are only some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

实施例1聚集发光光敏剂TTD的合成Example 1 Synthesis of aggregated luminescent photosensitizer TTD

聚集发光光敏剂TTD的合成步骤参考现有技术，具体合成路线如图2所示。The synthetic steps of the aggregated luminescent photosensitizer TTD refer to the prior art, and the specific synthetic route is shown in FIG. 2 .

实施例2 TTD/RIF@lipid-PEG多功能纳米胶束的制备Example 2 Preparation of TTD/RIF@lipid-PEG multifunctional nanomicelles

将20.0mg抗结核药物利福平(Rifampicin，RIF，美国sigma公司)溶解在1mL四氢呋喃(THF)溶液中，然后向其中加入1.0mg聚集发光光敏剂TTD和2.0mg双亲性胶束分子聚乙二醇-二硬脂酰基磷脂酰乙醇胺(DSPE-PEG2000，美国Avanti公司)，将其混合均匀后，快速注入到9mL的Milli-Q超纯水(美国Millipore公司)中，利用12W、25,000Hz的超声探头超声2min，之后将混合液倒入50ml的烧瓶中，加入磁子进行500rpm室温、避光搅拌过夜，除去混合液中的THF溶剂。利用0.22μm孔径的针头过滤器将大颗粒去除，然后利用10KDa超滤管超滤并洗涤多次，洗去所有未组装的游离RIF和DSPE-PEG2000，最终得到所述TTD/RIF@lipid-PEG多功能纳米胶束。20.0 mg of anti-tuberculosis drug rifampicin (Rifampicin, RIF, American sigma company) was dissolved in 1 mL of tetrahydrofuran (THF) solution, and then 1.0 mg of aggregated luminescent photosensitizer TTD and 2.0 mg of amphiphilic micellar molecule polyethylene glycol were added to it. Alcohol-distearoyl phosphatidylethanolamine (DSPE-PEG2000, Avanti, USA), after mixing it evenly, quickly poured it into 9 mL of Milli-Q ultrapure water (Millipore, USA), using 12W, 25,000Hz ultrasound The probe was sonicated for 2 min, then the mixture was poured into a 50ml flask, a magnet was added, and the mixture was stirred overnight at 500 rpm at room temperature in the dark, and the THF solvent in the mixture was removed. Use a 0.22 μm pore size syringe filter to remove large particles, and then use a 10KDa ultrafiltration tube to ultrafilter and wash multiple times to wash away all unassembled free RIF and DSPE-PEG2000, and finally obtain the TTD/RIF@lipid-PEG Multifunctional nanomicelles.

将制备得到的TTD/RIF@lipid-PEG多功能纳米胶束进行以下表征：The prepared TTD/RIF@lipid-PEG multifunctional nanomicelles were characterized as follows:

(1)透射电子显微镜(TEM)：透射电镜图像表示TTD/RIF@lipid-PEG纳米胶束的形貌和纳米尺寸，结果参照图3A，电镜图表示，在100nm的标尺下，TTD/RIF@lipid-PEG纳米胶束为尺寸均一的球型结构。(1) Transmission electron microscope (TEM): The TEM image shows the morphology and nanoscale of the TTD/RIF@lipid-PEG nanomicelles. The results are shown in Figure 3A. The lipid-PEG nanomicelles are spherical structures of uniform size.

(2)Zetasizer Nano纳米粒度/zeta电位检测仪：Zetasizer Nano纳米粒度/zeta电位检测仪图显示了TTD/RIF@lipid-PEG纳米胶束的水合粒径分布及zeta电位值，纳米粒度结果(图3B)表示，TTD/RIF@lipid-PEG纳米胶束的水合粒径约为100-120nm，结果与电镜结果一致；Zeta电位结果表示，其表面带负电，为-60mV左右，能在溶液中保持均一稳定的状态。(2) Zetasizer Nano nanoparticle size/zeta potential detector: Zetasizer Nano nanoparticle size/zeta potential detector diagram shows the hydrated particle size distribution and zeta potential value of TTD/RIF@lipid-PEG nanomicelles, and the results of nanoparticle size (Fig. 3B) shows that the hydrated particle size of TTD/RIF@lipid-PEG nanomicelles is about 100-120 nm, and the results are consistent with the electron microscopy results; the Zeta potential results show that the surface is negatively charged, about -60mV, and can be maintained in solution. uniform and stable state.

(3)紫外-可见分光光度计(UV-Vis)：紫外-可见吸收光谱，表示TTD/RIF@lipid-PEG纳米胶束的特征性吸收光谱。结果参照图4，TTD/RIF@lipid-PEG纳米胶束有2个特征性吸收光谱，分别为350nm和490nm。(3) UV-Vis spectrophotometer (UV-Vis): UV-Vis absorption spectrum, representing the characteristic absorption spectrum of TTD/RIF@lipid-PEG nanomicelles. Results Referring to Figure 4, the TTD/RIF@lipid-PEG nanomicelles had two characteristic absorption spectra at 350 nm and 490 nm, respectively.

(4)荧光分光光度计：荧光分光光谱，表示TTD/RIF@lipid-PEG纳米胶束的特征性发射光谱。结果参照图4，用488nm激发光激发TTD/RIF@lipid-PEG纳米胶束，致使其发射强烈的红色荧光，其最大发射波长为635nm。(4) Fluorescence spectrophotometer: Fluorescence spectroscopy, which represents the characteristic emission spectrum of TTD/RIF@lipid-PEG nanomicelles. Results Referring to Figure 4, TTD/RIF@lipid-PEG nanomicelles were excited with 488 nm excitation light, resulting in strong red fluorescence emission with a maximum emission wavelength of 635 nm.

(5)ABDA体外ROS检测试剂盒：ABDA(美国sigma公司)拥有3个特征性吸收峰，其峰值与浓度呈正比。当溶液中存在ROS氧自由基时，ABDA会被迅速降解，其3个吸收峰会下降。结果参照图5，用LED白光照射含有TTD/RIF@lipid-PEG纳米胶束的ABDA水溶液后，ABDA的3个特征性吸收峰会随时间梯度而下降，表明TTD/RIF@lipid-PEG纳米胶束在光照下能产生大量的ROS氧自由基。(5) ABDA in vitro ROS detection kit: ABDA (sigma company in the United States) has three characteristic absorption peaks, and the peak value is proportional to the concentration. When there are ROS oxygen radicals in the solution, ABDA will be rapidly degraded, and its three absorption peaks will drop. Results Referring to Figure 5, after irradiating the ABDA aqueous solution containing TTD/RIF@lipid-PEG nanomicelles with LED white light, the three characteristic absorption peaks of ABDA decreased with time gradient, indicating that TTD/RIF@lipid-PEG nanomicelles A large number of ROS oxygen free radicals can be generated under light.

(6)结核结节体内靶向成像能力：将TTD/RIF@lipid-PEG纳米胶束水溶液通过静脉注射到已构建了海分枝杆菌(Mycobacterium marinum，M.m)感染所致的斑马鱼局部结节模型和海分枝杆菌感染所致的小鼠尾部结节模型的动物体内后，经过一段时间的体内循环，用荧光显微镜和小动物荧光成像系统观察TTD/RIF@lipid-PEG纳米胶束对结节的靶向成像效果及在体内各器官的分布情况。结果参照图6，在成功构建局部结节的斑马鱼胚胎体内，静脉注射TTD/RIF@lipid-PEG纳米胶束后循环24h，通过倒置荧光显微镜观察，发现纳米胶束能够成功与结节共定位，表明其体内的结节靶向成像能力。结果参照图7，在成功构建尾部结节的小鼠体内，静脉注射TTD/RIF@lipid-PEG纳米胶束后循环12h，通过小动物荧光成像系统观察，发现纳米胶束能够成功与结节共定位，表明其体内的结节靶向成像能力；并且，TTD/RIF@lipid-PEG纳米胶束主要聚集于肝脏，而在其他脏器无聚集现象，表明其主要通过肝脏代谢。(6) In vivo targeting imaging capability of tuberculosis nodules: TTD/RIF@lipid-PEG nanomicelle aqueous solution was injected intravenously into local nodules in zebrafish infected with Mycobacterium marinum (M.m) After a period of in vivo circulation, the TTD/RIF@lipid-PEG nanomicelle pairs were observed with a fluorescence microscope and a small animal fluorescence imaging system. The targeted imaging effect of the node and its distribution in various organs in vivo. Results Referring to Figure 6, in the zebrafish embryos that successfully constructed local nodules, the TTD/RIF@lipid-PEG nanomicelles were intravenously injected and circulated for 24 h. Observed by an inverted fluorescence microscope, it was found that the nanomicelles could successfully co-localize with the nodules. , indicating its nodule-targeted imaging capabilities in vivo. Results Referring to Figure 7, in mice that successfully constructed tail nodules, TTD/RIF@lipid-PEG nanomicelles were injected intravenously and circulated for 12 hours. Through the observation of the small animal fluorescence imaging system, it was found that the nanomicelles could successfully co-exist with the nodules. The localization of TTD/RIF@lipid-PEG nanomicelles mainly aggregates in the liver, but no aggregation in other organs, indicating that it is mainly metabolized by the liver.

(7)结核菌的杀伤能力(7) The killing ability of Mycobacterium tuberculosis

利用不同浓度TTD/RIF@lipid-PEG纳米胶束与结核菌共孵育，并在白光照射30min后，利用CFU涂板计数判断其杀菌和抑菌能力。结果参照图8，在LED白光的照射下，与未光照组相比，随着TTD/RIF@lipid-PEG纳米胶束的浓度增加，结核菌的CFU明显下降，说明TTD/RIF@lipid-PEG纳米胶束对结核菌有光动力杀伤作用。并且，随着TTD/RIF@lipid-PEG纳米胶束的浓度增加，未光照组结核菌CFU也呈梯度下降，说明TTD/RIF@lipid-PEG纳米胶束内部包裹的抗结核药物RIF也发挥了抑菌作用。上述结果证明，TTD/RIF@lipid-PEG纳米胶束能够通过光动力、药物联合治疗更为有效的杀死结核杆菌并抑制结核菌的增殖。Different concentrations of TTD/RIF@lipid-PEG nanomicelles were used to co-incubate with Mycobacterium tuberculosis, and after 30 min of white light irradiation, the sterilization and bacteriostatic abilities were judged by CFU plate count. Results Referring to Figure 8, under the irradiation of LED white light, compared with the unirradiated group, with the increase of the concentration of TTD/RIF@lipid-PEG nanomicelles, the CFU of Mycobacterium tuberculosis decreased significantly, indicating that TTD/RIF@lipid-PEG Nanomicelles have photodynamic killing effect on Mycobacterium tuberculosis. Moreover, with the increase of the concentration of TTD/RIF@lipid-PEG nanomicelles, the CFU of Mycobacterium tuberculosis in the unirradiated group also decreased gradually, indicating that the anti-tuberculosis drug RIF encapsulated inside the TTD/RIF@lipid-PEG nanomicelles also played a role. Bacteriostatic effect. The above results prove that TTD/RIF@lipid-PEG nanomicelles can kill Mycobacterium tuberculosis more effectively and inhibit the proliferation of Mycobacterium tuberculosis by photodynamic and drug combination therapy.

Claims

1. The multifunctional nano-micelle is used for targeted imaging of tuberculosis and multi-modal therapy, and is characterized in that the multifunctional nano-micelle is of a core-shell structure, the core of the multifunctional nano-micelle is composed of an aggregated luminescent photosensitizer TTD and an anti-tuberculosis drug, the shell is a monolayer lipid membrane, and the diameter of the multifunctional nano-micelle is 100-120 nm; the anti-tuberculosis drug is rifampicin.

2. The multifunctional nanomicelle for tuberculosis targeted imaging and multimodal therapy according to claim 1, characterized in that the unilamellar lipid membrane is aggregated from amphiphilic micelle molecule polyethylene glycol 2000-distearoylphosphatidylethanolamine.

3. The method for preparing multifunctional nanomicelle according to claim 2, comprising the steps of:

(1) dissolving rifampicin in THF solvent, and adding aggregated luminescent photosensitizer TTD and amphiphilic micelle molecule polyethylene glycol 2000-distearoyl phosphatidyl ethanolamine to form THF mixed solution; wherein the mass ratio of the aggregated luminescent photosensitizer TTD to rifampicin to the amphiphilic micelle molecule polyethylene glycol 2000-distearoylphosphatidylethanolamine is 1-2: 12-30: 2-4.

(2) Injecting the THF mixed solution into ultrapure water, performing ultrasonic and photophobic magnetic stirring, and evaporating the THF solvent to dryness;

(3) and filtering for multiple times, and washing away the unbound rifampicin and amphiphilic micelle molecule polyethylene glycol 2000-distearoylphosphatidylethanolamine to obtain the TTD/RIF @ lipid-PEG multifunctional nano-micelle.

4. The preparation method of multifunctional nano-micelle of claim 3, wherein the conditions of the ultrasound in the step (2) are 60-80W, 20-25kHz, and the time is 1-5 min.

5. The method for preparing multifunctional nanomicelle according to claim 3, wherein the multiple filtration of step (3) is performed by a needle filter with a pore size of 0.22 μm and then by ultrafiltration with ultrafiltration of 10 kDa.

6. A formulation for use in the treatment of tuberculosis, characterized in that it comprises the multifunctional nanomicelle according to any one of claims 1 to 2.

7. Use of the multifunctional nanomicelle according to any one of claims 1 to 2 for the preparation of a contrast agent for imaging of tuberculosis lesions.

8. Use of the multifunctional nanomicelle according to any one of claims 1 to 2 for the preparation of a photodynamic, drug co-biocide for tubercle bacillus.