(一)技术领域(1) Technical field
本发明涉及一种用于肿瘤早期诊断与光学治疗的Pdots/Ce6lipid-Gd-DOTA多功能纳米胶束及其应用。The invention relates to a Pdots/Ce6lipid-Gd-DOTA multifunctional nano-micelle for early tumor diagnosis and optical therapy and application thereof.
(二)背景技术(2) Background technology
肝细胞癌是消化系统中常见的恶性肿瘤之一，具有发展速度快、恶性程度高和预后差等特点，其发病率呈逐年上升的趋势。作为高发性恶性肿瘤,肝癌在福建省癌症死亡率居所有恶性肿瘤之首,约每10万人中32人死于肝癌。目前肝癌的治疗仍以手术切除为主，但很多患者在确诊时因肿瘤部位，身体耐受状态及肝硬化程度的影响而失去手术治疗机会。尽管局部化疗、介入治疗、射频消融治疗、免疫治疗甚至肝移植等方式均被广泛应用于肝癌的综合治疗，并取得了显著的疗效，但肝癌病人的远期生存率仍不够理想(5年生存率不足8％，5年复发转移率更是高达50-70％)。因此，急需发展新的诊断和治疗模式来进一步提高肝癌的远期生存率。近年来发展起来的“诊断治疗学”，将诊断和治疗整合于同一纳米探针上，同时实现肿瘤的高灵敏度检测与微创治疗，其优势在于操作简便、成像灵敏度更高、诊断和治疗同时进行等，为肝癌的“早诊早治”提供了新的思路和手段。Hepatocellular carcinoma is one of the common malignant tumors in the digestive system. It has the characteristics of rapid development, high degree of malignancy and poor prognosis, and its incidence is increasing year by year. As a high-incidence malignant tumor, the cancer mortality rate of liver cancer ranks first among all malignant tumors in Fujian Province, about 32 people per 100,000 people died of liver cancer. At present, the treatment of liver cancer is still based on surgical resection, but many patients lose the chance of surgical treatment due to the influence of tumor location, physical tolerance status and degree of liver cirrhosis when they are diagnosed. Although local chemotherapy, interventional therapy, radiofrequency ablation therapy, immunotherapy and even liver transplantation have been widely used in the comprehensive treatment of liver cancer and have achieved remarkable curative effects, the long-term survival rate of liver cancer patients is still not ideal (5-year survival rate is less than 8%, and the 5-year recurrence and metastasis rate is as high as 50-70%). Therefore, there is an urgent need to develop new diagnostic and therapeutic models to further improve the long-term survival rate of liver cancer. The "diagnostics and therapeutics" developed in recent years integrates diagnosis and treatment on the same nanoprobe, and realizes high-sensitivity detection and minimally invasive treatment of tumors at the same time. Its advantages lie in easy operation, higher imaging sensitivity, and simultaneous diagnosis and treatment. It provides new ideas and means for the "early diagnosis and early treatment" of liver cancer.
磁共振成像是利用人体组织中氢原子核(质子)在磁场中受到射频脉冲的激励而产生磁共振信号，经电子计算机处理,可在亚毫米水平上提供高分辨率的组织信息和三维结构成像，对肝癌的诊断优于CT成像。但高对比度的获取较多依赖于造影剂的介入或灌注。而目前商用造影剂体内清除太快、无特异性分布等因素，导致对比效果不明显。Magnetic resonance imaging uses hydrogen nuclei (protons) in human tissue to be excited by radio frequency pulses in a magnetic field to generate magnetic resonance signals. After computer processing, it can provide high-resolution tissue information and three-dimensional structural imaging at the submillimeter level. The diagnosis of liver cancer is better than CT imaging. However, the acquisition of high contrast depends more on the intervention or perfusion of contrast agents. However, the current commercial contrast agents are cleared too quickly in the body, have no specific distribution and other factors, resulting in insignificant contrast effects.
近年来，基于光声效应的光声成像技术是快速发展的非侵入式、非电离的新型生物医学影像技术。它继承了光学成像与超声成像的技术特点，具备光学成像的高对比度与超声成像的高分辨率的特性，能够从形态与功能成像两方面同时对生物组织体进行成像。光声信号与光声图像所承载的大量信息，均与组织体的结构、成份、功能息息相关，而分析与研究这部分信息，将能够以无损的方式下实现对组织体的生理与病理的检测。然而，光声成像虽然具有诸多成像优势，但光声成像的光源对组织的穿透深度有一定的局限性。随着纳米技术的不断发展，将多种功能附加于同一纳米材料上已成为可能，而以优势互补的方式将磁共振与光声成像技术加以整合，将更有助于提高临床对癌症的检出率。In recent years, photoacoustic imaging technology based on photoacoustic effect is a rapidly developing non-invasive, non-ionizing new biomedical imaging technology. It inherits the technical characteristics of optical imaging and ultrasonic imaging, has the characteristics of high contrast of optical imaging and high resolution of ultrasonic imaging, and can simultaneously image biological tissues from both morphological and functional imaging. A large amount of information carried by photoacoustic signals and photoacoustic images is closely related to the structure, composition, and function of the tissue, and the analysis and research of this part of the information will enable the detection of the physiology and pathology of the tissue in a non-destructive manner. . However, although photoacoustic imaging has many imaging advantages, the light source of photoacoustic imaging has certain limitations in the penetration depth of tissues. With the continuous development of nanotechnology, it has become possible to add multiple functions to the same nanomaterial, and the integration of magnetic resonance and photoacoustic imaging in a complementary manner will help improve the clinical detection of cancer. out rate.
光动力治疗与光热治疗是完全不同于手术治疗和化疗的肿瘤新疗法，已成为肿瘤防治研究的热点。光动力治疗是以光源、光敏剂、氧气三者相互作用为基础的光敏化治疗方式，其产生的强氧化活性的单线态氧(1O2)，破坏胞内DNA、脂类等生物活性物质，诱导细胞凋亡；光热治疗是利用近红外激光照射生物体的治疗部位，促使胞内外源性光热转换物质吸收光能并转化为高热能，使局部温度升高到46℃以上，引发肿瘤组织坏死，达到肿瘤治疗的目的。但目前采用的光敏剂或光热转换材料均存在毒性较大、疏水性较强、体内代谢缓慢、易被网状内皮系统清除等问题，不利于光动力、光热治疗在临床上的进一步应用。Photodynamic therapy and photothermal therapy are new tumor treatments that are completely different from surgery and chemotherapy, and have become a hot spot in cancer prevention and treatment research. Photodynamic therapy is a photosensitization treatment method based on the interaction of light source, photosensitizer, and oxygen. The singlet oxygen (1 O2 ) with strong oxidative activity produced by it can destroy biologically active substances such as intracellular DNA and lipids. , to induce cell apoptosis; photothermal therapy is to use near-infrared laser to irradiate the treatment part of the organism, to promote the exogenous photothermal conversion substances in the cells to absorb light energy and convert it into high heat energy, so that the local temperature rises above 46 ° C, triggering Tumor tissue necrosis achieves the purpose of tumor treatment. However, the photosensitizers or photothermal conversion materials currently used have problems such as high toxicity, strong hydrophobicity, slow metabolism in the body, and easy removal by the reticuloendothelial system, which is not conducive to the further clinical application of photodynamic and photothermal therapy. .
(三)发明内容(3) Contents of the invention
本发明目的是提供一种适用于癌症早期诊断的核磁成像(MRI)，光声成像，近红外荧光成像以及光热、光动力联合治疗的Pdots/Ce6lipid-Gd-DOTA多功能纳米胶束及其应用。The object of the present invention is to provide a kind of nuclear magnetic imaging (MRI) that is applicable to the early diagnosis of cancer, photoacoustic imaging, near-infrared fluorescence imaging and photothermal, photodynamic combined therapy Pdots/Ce6lipid-Gd-DOTA multifunctional nano-micelle and its application.
本发明采用的技术方案是：The technical scheme adopted in the present invention is:
一种用于肿瘤早期诊断与光学治疗的Pdots/Ce6lipid-Gd-DOTA多功能纳米胶束，由单层脂质膜外壳与其内部包埋的光敏剂与光热吸收剂组成，直径约为36～50nm；所述的单层脂质膜为表面修饰螯合有Gd3+离子的DOTA耦合剂的聚乙二醇-二硬脂酰基磷脂酰乙醇胺(DSPE-PEG)脂质体膜，所述光敏剂为二氢卟吩e6(Chlorin e6，Ce6)，所述光热吸收剂为聚[2,1,3-苯并噻二唑-4,7-二基[4,4-双(2-乙基己基)-4H-环戊并[2,1-b:3,4-b']二噻吩-2,6-二基]](PCPDTBT)有机半导体聚合物经超声波法而形成的粒径为3～4nm的有机半导体量子点(Pdots)。A Pdots/Ce6lipid-Gd-DOTA multifunctional nanomicelle for early tumor diagnosis and optical therapy, which is composed of a single-layer lipid membrane shell and a photosensitizer and a photothermal absorber embedded in it, with a diameter of about 36~ 50nm; Described monolayer lipid membrane is the polyethylene glycol-distearoyl phosphatidylethanolamine (DSPE-PEG) liposome membrane of the DOTA coupling agent that surface modification chelates Gd3+ ions, and the photosensitive The agent is chlorin e6 (Chlorin e6, Ce6), and the photothermal absorber is poly[2,1,3-benzothiadiazole-4,7-diyl[4,4-bis(2- Particle size of ethylhexyl)-4H-cyclopenta[2,1-b:3,4-b']dithiophene-2,6-diyl]](PCPDTBT) organic semiconductor polymer formed by ultrasonic method 3 ~ 4nm organic semiconductor quantum dots (Pdots).
本发明在聚乙二醇-二硬脂酰基磷脂酰乙醇胺(DSPE-PEG)表面修饰螯合有Gd3+离子的DOTA耦合剂，在一定的磁场作用下具有MRI造影功能，而Chlorin e6为一种带负电的近红外光敏剂，可被670nm的近红外激光激发，并与分子氧作用，产生氧自由基(ROS)用于诱导癌细胞凋亡；而聚[2,1,3-苯并噻二唑-4,7-二基[4,4-双(2-乙基己基)-4H-环戊并[2,1-b:3,4-b']二噻吩-2,6-二基]](PCPDTBT)有机半导体聚合物在超声波法作用下，可形成粒径为3～4nm的有机半导体量子点(Pdots)，其近红外吸收波长在650nm～750nm之间，同时在利用670nm近红外激光激发下，能够将光能转变为过高热能，提高肿瘤区组织的温度，用于热诱导癌细胞坏死。The DOTA coupling agent with Gd3+ ions modified and chelated on the surface of polyethylene glycol-distearoylphosphatidylethanolamine (DSPE-PEG) of the present invention has MRI imaging function under the action of a certain magnetic field, and Chlorin e6 is a A negatively charged near-infrared photosensitizer, which can be excited by a 670nm near-infrared laser, and react with molecular oxygen to generate oxygen free radicals (ROS) for inducing cancer cell apoptosis; while poly[2,1,3-benzo Thiadiazole-4,7-diyl[4,4-bis(2-ethylhexyl)-4H-cyclopenta[2,1-b:3,4-b']dithiophene-2,6- Dibasic]] (PCPDTBT) organic semiconductor polymer can form organic semiconductor quantum dots (Pdots) with a particle size of 3-4nm under the action of ultrasonic method, and its near-infrared absorption wavelength is between 650nm and 750nm. Under the excitation of near-infrared laser, it can convert light energy into superheat energy, increase the temperature of tumor tissue, and be used for heat-induced necrosis of cancer cells.
上述构建的Pdots/Ce6lipid-Gd-DOTA多功能纳米胶束具有较高的生物相容性与光热稳定性，具备可生物降解与生物利用率高等特点。而纳米化的Gd耦合剂具有更高的弛豫率(高于医用钆普胺)，以及血液清除缓慢等优势。因此，可有效提高磁共振成像对比度，延长磁共振显像时间；同时，在670nm的脉冲激光激发下，Pdots/Ce6lipid-Gd-DOTA多功能纳米胶束良好的热声性能，可用于对肿瘤组织进行光声成像，用于癌症的早期诊断；在670nm近红外光照射下，内部包埋的Chlorin e6分子与Pdots能够有效的将光能分别转化为化学能与热能，产生大量的ROS与50～60℃的过高热，进而在氧自由基诱导癌细胞凋亡的同时协同过高热杀伤癌细胞。The Pdots/Ce6lipid-Gd-DOTA multifunctional nanomicelle constructed above has high biocompatibility and photothermal stability, and has the characteristics of biodegradability and high bioavailability. The nano-sized Gd coupling agent has the advantages of higher relaxation rate (higher than medical gadopromide) and slow blood clearance. Therefore, it can effectively improve the contrast of magnetic resonance imaging and prolong the time of magnetic resonance imaging; at the same time, under the excitation of 670nm pulsed laser, the good thermoacoustic properties of Pdots/Ce6lipid-Gd-DOTA multifunctional nanomicelles can be used to treat tumor tissue Perform photoacoustic imaging for early diagnosis of cancer; under the irradiation of 670nm near-infrared light, the embedded Chlorin e6 molecules and Pdots can effectively convert light energy into chemical energy and heat energy respectively, and generate a large amount of ROS and 50~ Hyperthermia at 60°C, and then cooperate with hyperthermia to kill cancer cells while oxygen free radicals induce cancer cell apoptosis.
为提高纳米胶束的体内循环时间，降低网状内皮系统(Reticuloendothelial system)的吞噬作用，本发明采用FDA批准的、已用于临床的DSPE-PEG(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。本发明为提高癌症的早期诊断，弥补传统成像手段对肿瘤组织成像对比度低与软组织定性诊断困难等问题，利用Gd螯合剂功能化的DSPE-PEG提高磁共振成像的对比度，利用新颖的光声成像提高对肿瘤组织的功能性成像检测。因此，本发明的脂质体颗粒外壳除了具有对肝癌细胞MRI成像功能，还具备良好的生物相容性。For improving the in vivo circulation time of nano-micelle, reduce the phagocytosis of reticuloendothelial system (Reticuloendothelial system), the present invention adopts the DSPE-PEG (polyethylene glycol-distearoyl phosphatidyl glycol-distearoyl phosphatidyl) that has been used clinically. ethanolamine) liposome membrane, which can effectively improve the drug body circulation time. In order to improve the early diagnosis of cancer, the present invention makes up for the low contrast of tumor tissue imaging by traditional imaging means and the difficulty of qualitative diagnosis of soft tissue, etc., using Gd chelating agent functionalized DSPE-PEG to improve the contrast of magnetic resonance imaging, and using novel photoacoustic imaging Improve functional imaging detection of tumor tissue. Therefore, the liposome particle shell of the present invention not only has the MRI imaging function of liver cancer cells, but also has good biocompatibility.
本发明构建的Pdots/Ce6lipid-Gd-DOTA多功能纳米胶束，同时具备对肿瘤的核磁成像、光声成像与近红外荧光成像等多模态影像的功能，提高MRI的T1加权成像信号强度，提高对肿瘤组织的功能性与光声成像的分辨率，并且兼有对肿瘤的光热治疗与光动力治疗协同治疗功能。The Pdots/Ce6lipid-Gd-DOTA multifunctional nanomicelle constructed by the present invention has the functions of nuclear magnetic imaging, photoacoustic imaging and near-infrared fluorescence imaging of tumors at the same time, and improves the T1 weighted imaging signal intensityof MRI. , improve the functionality of tumor tissue and the resolution of photoacoustic imaging, and also have the function of synergistic treatment of tumor photothermal therapy and photodynamic therapy.
具体的，所述多功能纳米胶束由如下方法制得：Specifically, the multifunctional nanomicelle is prepared by the following method:
(1)由DSPE-PEG-NH2和DOTA-NHS(1,4,7,10-四乙酸-N-羟基琥珀酰亚胺)进行酰胺偶联反应，并耦合Gd3+离子，得到DSPE-PEG-Gd-DOTA(二硬脂酰基磷脂酰乙醇胺-聚乙二醇-1,4,7,10-四乙酸钆)；该步骤可得到DSPE-PEG-Gd-DOTA冻干粉；(1) Carry out amide coupling reaction by DSPE-PEG-NH2 and DOTA-NHS (1,4,7,10-tetraacetic acid-N-hydroxysuccinimide), and couple Gd3+ ions to obtain DSPE- PEG-Gd-DOTA (distearoylphosphatidylethanolamine-polyethylene glycol-1,4,7,10-gadolinium tetraacetate); this step can obtain DSPE-PEG-Gd-DOTA freeze-dried powder;
具体方法如下：DSPE-PEG-NH2与DOTA-NHS在水溶液中，室温反应2h，转移到4℃反应12～24h，后用5KDa的超滤管离心后获得DSPE-PEG-DOTA，之后在水溶液中加入GdCl3粉末，强烈震荡48h后，利用5KDa超滤管离心，获得DSPE-PEG-Gd-DOTA；然后，利用低温冷冻干燥器将DSPE-PEG-Gd-DOTA冻干后备用；The specific method is as follows: DSPE-PEG-NH2 and DOTA-NHS were reacted in aqueous solution for 2 hours at room temperature, transferred to 4°C for 12-24 hours, and then centrifuged with a 5KDa ultrafiltration tube to obtain DSPE-PEG-DOTA, and then dissolved in aqueous solution GdCl3 powder was added to the mixture, shaken vigorously for 48 hours, and centrifuged with a 5KDa ultrafiltration tube to obtain DSPE-PEG-Gd-DOTA; then, DSPE-PEG-Gd-DOTA was freeze-dried using a low-temperature freeze dryer for later use;
(2)将聚[2,1,3-苯并噻二唑-4,7-二基[4,4-双(2-乙基己基)-4H-环戊并[2,1-b:3,4-b']二噻吩-2,6-二基]](PCPDTBT)溶解在1mL的四氢呋喃(THF)溶液中，然后向其中加入光敏剂二氢卟吩e6(Chlorine6)，氢化大豆磷脂(HSPC)和步骤(1)中制备的DSPE-PEG-Gd-DOTA白色粉末，均衡混合后，将所得THF混合液注入到10mL去离子水中，用40W的超声探头超声3min，然后，将得到的混合液投入到50mL的圆底烧瓶中，在500rpm/min的室温、避光条件下搅拌24h，去除混合液中的THF溶剂；之后，将混合液置于10KDa的透析袋中进行透析，3天，最终，得到所述Pdots/Ce6lipid-Gd-DOTA多功能纳米胶束；所述PCPDTBT、Chlorin e6和HSPC、DSPE-PEG-Gd-DOTA的质量之比为：2～4：1～2：5～10：2～4。(2) Poly[2,1,3-benzothiadiazole-4,7-diyl[4,4-bis(2-ethylhexyl)-4H-cyclopenta[2,1-b: 3,4-b']dithiophene-2,6-diyl]](PCPDTBT) was dissolved in 1 mL of tetrahydrofuran (THF) solution, and then the photosensitizer chlorin e6 (Chlorine6), a hydrogenated soybean lecithin, was added to it (HSPC) and the DSPE-PEG-Gd-DOTA white powder prepared in step (1), after balanced mixing, inject the gained THF mixed solution into 10mL deionized water, ultrasonic 3min with the ultrasonic probe of 40W, then, the obtained Put the mixed solution into a 50mL round-bottomed flask, stir at 500rpm/min at room temperature for 24h in the dark, and remove the THF solvent in the mixed solution; after that, place the mixed solution in a 10KDa dialysis bag for dialysis for 3 days , finally, obtain the Pdots/Ce6lipid-Gd-DOTA multifunctional nano-micelle; the mass ratio of the PCPDTBT, Chlorine6 and HSPC, DSPE-PEG-Gd-DOTA is: 2～4:1～2:5 ～10: 2～4.
DSPE-PEG-Gd-DOTA、PCPDTBT和氢化大豆磷脂、Ce6的质量之比优选为2:1:5:2。The mass ratio of DSPE-PEG-Gd-DOTA, PCPDTBT, hydrogenated soybean lecithin and Ce6 is preferably 2:1:5:2.
本发明还涉及所述的Pdots/Ce6lipid-Gd-DOTA多功能纳米胶束在制备用于肿瘤的多模态成像的造影剂中的应用。所述造影剂可用于MRI磁共振成像，光声成像或近红外荧光成像。The present invention also relates to the application of the Pdots/Ce6lipid-Gd-DOTA multifunctional nano-micelle in the preparation of a contrast agent for multimodal imaging of tumors. The contrast agent can be used for MRI magnetic resonance imaging, photoacoustic imaging or near-infrared fluorescence imaging.
本发明还涉及所述的Pdots/Ce6lipid-Gd-DOTA多功能纳米胶束在制备用于肿瘤的光动力、光热治疗剂中的应用。所述的Pdots/Ce6lipid-Gd-DOTA多功能纳米胶束，在单一近红外激光(670nm)的照射下，同时发挥对肿瘤的光动力治疗、光热治疗协同治疗应用。The present invention also relates to the application of the Pdots/Ce6lipid-Gd-DOTA multifunctional nano micelles in the preparation of photodynamic and photothermal therapeutic agents for tumors. The Pdots/Ce6lipid-Gd-DOTA multifunctional nanomicelle, under the irradiation of a single near-infrared laser (670nm), simultaneously exerts the synergistic therapeutic application of photodynamic therapy and photothermal therapy on tumors.
本发明以有机半导体量子点Pdots与光敏剂Chlorin e6为共载体，形成纳米胶束的核心，以独特的设计方案，将耦合有Gd-DOTA的脂质体膜，用于包埋Pdots与Chlorin e6，并设计合成Pdots/Ce6lipid-Gd-DOTA纳米胶束，获得具有多模态成像及单一激光激发同时发挥光动力、光热治疗功能的多功能纳米胶束。该Pdots/Ce6lipid-Gd-DOTA纳米胶束具有36-50nm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 present invention, organic semiconductor quantum dots Pdots and photosensitizer Chlorine e6 are used as co-carriers to form the core of nano micelles, and a liposome membrane coupled with Gd-DOTA is used to embed Pdots and Chlorine e6 with a unique design scheme. , and designed and synthesized Pdots/Ce6lipid-Gd-DOTA nanomicelles to obtain multifunctional nanomicelles with multi-modal imaging and single laser excitation while exerting photodynamic and photothermal therapeutic functions. The Pdots/Ce6lipid-Gd-DOTA nanomicelle has a diameter of 36-50 nm, which is equivalent to the size of some proteins and bioorganic compounds present in human and animal organisms, thereby facilitating the delivery and absorption of contrast agents in areas of interest .
本发明所构建的Pdots/Ce6lipid-Gd-DOTA多功能纳米胶束可通过不同的剂量给药，这取决于早期成像诊断的癌细胞，组织及癌症患者的临床状况。本发明的造影剂和光动力、光热药物的给药剂量和浓度，可由临床医生例行确定。用药方案取决于各种因素，例如成像的癌细胞，组织或肿瘤是分散还是局部，患者的健康程度，年龄等。通过参照其他造影剂的用药方案，本领域技术人员能够确定本发明药物的最佳有效剂量及浓度。The Pdots/Ce6lipid-Gd-DOTA multifunctional nanomicelle constructed in the present invention can be administered in different doses, depending on the cancer cells, tissues and clinical conditions of cancer patients diagnosed by early imaging. The doses and concentrations of the contrast agent and photodynamic and photothermal drugs of the present invention can be routinely determined by clinicians. The dosing regimen depends on various factors, such as the cancer cells being imaged, whether the tissue or tumor is diffuse or localized, the patient's health, age, and more. Those skilled in the art can determine the optimal effective dosage and concentration of the drug of the present invention by referring to the dosage regimens of other contrast agents.
本发明的所构建的Pdots/Ce6lipid-Gd-DOTA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，肿瘤内，胃肠外，肺腔内，局部、或局部给药的区域递送形式。如动脉内注射能够被用来“局域效果”成像及特定区域的光动力、光热治疗。The constructed Pdots/Ce6lipid-Gd-DOTA multifunctional nanomicelles of the present invention can be administered by any known delivery method: systemic delivery (intravenous injection), intraarterial, intratumoral, parenteral, and intrapulmonary cavity , a topical, or regional delivery form for topical administration. For example, intra-arterial injection can be used for "local effect" imaging and photodynamic and photothermal therapy of specific regions.
本发明Pdots/Ce6lipid-Gd-DOTA多功能纳米胶束可用于肝癌的核磁成像(MRI)，光声成像，近红外荧光成像(Near-infrared(NIR)fluorescence imaging)等多种影像模式，只需承受一种造影剂给药，即可达到多种诊断效果；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670nm近红外光照射下，内部包埋的Pdots和Chlorine6光敏分子，能够有效的将光能转化为热能或化学能，产生60～70℃的过高热或大量ROS氧自由基，进而用于杀伤肿瘤细胞；因此，该多功能纳米胶束不仅可用于癌症的多模态成像，还可作为肿瘤的光动力、光热协同治疗应用。The Pdots/Ce6lipid-Gd-DOTA multifunctional nano-micelle of the present invention can be used in multiple imaging modes such as nuclear magnetic imaging (MRI), photoacoustic imaging, and near-infrared (NIR) fluorescence imaging of liver cancer. A variety of diagnostic effects can be achieved by accepting the administration of one contrast agent; and due to the improvement of sensitivity, the dosage of contrast agent can be reduced, and the toxic and side effects can be further reduced. Under the irradiation of 670nm near-infrared light, the embedded Pdots and Chlorine6 photosensitive molecules can effectively convert light energy into thermal energy or chemical energy, and generate excessive heat at 60-70°C or a large number of ROS oxygen free radicals, which are then used to kill Tumor cells; therefore, the multifunctional nanomicelles can be used not only for multimodal imaging of cancer, but also for photodynamic and photothermal synergistic therapy of tumors.
(四)附图说明(4) Description of drawings
图1为Pdots/Ce6lipid-Gd-DOTA多功能纳米胶束的透射电镜图像(200nm标尺)；Fig. 1 is the transmission electron microscope image (200nm scale) of Pdots/Ce6lipid-Gd-DOTA multifunctional nanomicelle;
图2为Pdotslipid micelles，Ce6，Pdots/Ce6lipid-Gd-DOTA micelles的紫外可见近红外吸收光谱；(—)Pdots/Ce6lipid-Gd-DOTA micelles，(--)Ce6，(-·-)表示Pdotslipid micelles的紫外可见近红外吸收光谱曲线图；Figure 2 is the ultraviolet-visible-near-infrared absorption spectrum of Pdotslipid micelles, Ce6, Pdots/Ce6lipid-Gd-DOTA micelles; (—) Pdots/Ce6lipid-Gd-DOTA micelles, (--) Ce6, (-·-) means Pdotslipid micelles The UV-Vis-NIR absorption spectrum curve;
图3为Pdots/Ce6lipid-Gd-DOTA多功能纳米胶束的T1磁共振加权成像图(A)和Pdots/Ce6lipid-Gd-DOTA多功能纳米胶束的光声成像图(B)；图4为Pdots/Ce6lipid-Gd-DOTA，Ce6，Pdotslipid micelles分别对肝癌细胞的光热-光动力联合杀伤、光动力杀伤、光热杀伤。Fig.3 is the T1 magnetic resonance weighted imaging picture (A) of Pdots/Ce6lipid-Gd-DOTA multifunctional nanomicelle and the photoacoustic imaging picture (B) of Pdots/Ce6lipid-Gd-DOTA multifunctional nanomicelle; Fig. 4 Pdots/Ce6lipid-Gd-DOTA, Ce6, Pdotslipid micelles respectively on liver cancer cell photothermal-photodynamic combined killing, photodynamic killing, photothermal killing.
(五)具体实施方式(5) Specific implementation methods
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:
实施例1：Example 1:
利用电子天平称取4.2mg的DSPE-PEG-NH2(聚乙二醇-二硬脂酰基磷脂酰乙醇胺，Nanocs公司)白色粉末和4.2mg的DOTA-NHS白色粉末(钆离子螯合剂，Macrocyclic公司)白色粉末，溶解在盛有1ml水溶液的1.5ml的离心管中，室温震荡反应2h后，转移到4℃冰箱，震荡反应24h，后用5KDa的超滤管过滤，收集DSPE-PEG-DOTA-NHS，之后向其中加入60mg的GdCl3(三氯化钆，百灵威试剂公司)白色粉末，强烈震荡12h后，利用5KDa的超滤管过滤，收集DSPE-PEG-Gd-DOTA，并利用低温冷冻干燥器将其冻干备用。Utilize the electronic balance to weigh 4.2mg of DSPE-PEG-NH2 (polyethylene glycol-distearoyl phosphatidylethanolamine, Nanocs company) white powder and 4.2mg of DOTA-NHS white powder (gadolinium ion chelating agent, Macrocyclic company) ) white powder, dissolved in a 1.5ml centrifuge tube filled with 1ml of aqueous solution, after shaking at room temperature for 2 hours, transferred to a refrigerator at 4°C, shaking for 24 hours, and then filtered with a 5KDa ultrafiltration tube to collect DSPE-PEG-DOTA- Then add 60 mg of GdCl3 (gadolinium trichloride, Bailingwei Reagent Co., Ltd.) white powder to the NHS, shake it vigorously for 12 hours, filter it with a 5KDa ultrafiltration tube, collect DSPE-PEG-Gd-DOTA, and use low-temperature freeze-drying Freeze-dry it for later use.
实施例2：Example 2:
将0.25mg的聚[2,1,3-苯并噻二唑-4,7-二基[4,4-双(2-乙基己基)-4H-环戊并[2,1-b:3,4-b']二噻吩-2,6-二基]](PCPDTBT)(美国sigma公司)溶于1mL的THF中，并向其加入0.5mg的二氢卟吩e6(Chlorin e6)粉末、2.5mg的氢化大豆磷脂(HSPC，上海艾韦特医药科技有限公司)、0.5mg的DSPE-PEG-Gd-DOTA粉末，将其混合均匀后，快速注入10mL的Milli-Q超纯水(美国Millipore公司)中，利用40W的超声探头进行超声处理3min，之后将混合液倒入50ml的烧瓶中，加入磁子进行500rpm下，室温、避光搅拌过夜，除去混合液中的THF溶剂。之后利用10KDa的透析袋透析3天，除去多余的Chlorin e6分子和Pdots，得到Pdots/Ce6lipid-Gd-DOTA多功能纳米胶束。0.25 mg of poly[2,1,3-benzothiadiazole-4,7-diyl[4,4-bis(2-ethylhexyl)-4H-cyclopenta[2,1-b: 3,4-b']dithiophene-2,6-diyl]](PCPDTBT) (Sigma, USA) was dissolved in 1 mL of THF, and 0.5 mg of chlorin e6 (Chlorin e6) powder was added to it , 2.5 mg of hydrogenated soybean lecithin (HSPC, Shanghai Avete Pharmaceutical Technology Co., Ltd.), 0.5 mg of DSPE-PEG-Gd-DOTA powder, after mixing them evenly, quickly inject 10 mL of Milli-Q ultrapure water (USA Millipore Company), utilize the ultrasonic probe of 40W to carry out sonication 3min, then the mixed solution is poured in the flask of 50ml, add magneton and carry out under 500rpm, room temperature, dark stirring overnight, remove the THF solvent in the mixed solution. Afterwards, dialysis was performed for 3 days using a 10KDa dialysis bag to remove excess Chlorine e6 molecules and Pdots to obtain Pdots/Ce6lipid-Gd-DOTA multifunctional nanomicelles.
实施例3：Example 3:
实施例2制备的Pdots/Ce6lipid-Gd-DOTA纳米胶束的粒子直径约36±12nm，并具备了MRI、光声成像、近红外成像，光动力、光热治疗功能。The particle diameter of the Pdots/Ce6lipid-Gd-DOTA nanomicelle prepared in Example 2 is about 36±12nm, and has the functions of MRI, photoacoustic imaging, near-infrared imaging, photodynamic and photothermal therapy.
使用透射电子显微镜(TEM)、紫外－可见分光光度计(UV-Vis-NIR)，磁共振成像系统，光声成像系统，对Pdots/Ce6lipid-Gd-DOTA纳米胶束进行表征，并利用670nm近红外激光分别照射Pdots/Ce6lipid-Gd-DOTA纳米胶束，Ce6，Pdotslipid纳米胶束的肝癌细胞，用于鉴定其光动力、光热联合杀伤的优势与效果。具体测试结果如下：Using transmission electron microscope (TEM), ultraviolet-visible spectrophotometer (UV-Vis-NIR), magnetic resonance imaging system, photoacoustic imaging system, the Pdots/Ce6lipid-Gd-DOTA nanomicelles were characterized, and 670nm near Infrared laser irradiation of Pdots/Ce6lipid-Gd-DOTA nanomicelles, Ce6, Pdotslipid nanomicelles of liver cancer cells was used to identify the advantages and effects of photodynamic and photothermal combined killing. The specific test results are as follows:
(1)透射电子显微镜(TEM)(1) Transmission Electron Microscope (TEM)
透射电镜图像表示Pdots/Ce6lipid-Gd-DOTA纳米胶束的形貌和纳米尺寸。结果参照图1，表示在200nm的标尺下，检测Pdots/Ce6lipid-Gd-DOTA纳米胶束的分布及形貌，其说明Pdots/Ce6lipid-Gd-DOTA纳米胶束尺寸均一。TEM images showing the morphology and nanometer size of Pdots/Ce6lipid-Gd-DOTA nanomicelles. The results refer to Figure 1, which shows the distribution and morphology of Pdots/Ce6lipid-Gd-DOTA nanomicelles detected under the scale of 200nm, which shows that the size of Pdots/Ce6lipid-Gd-DOTA nanomicelles is uniform.
(2)紫外-可见-近红外分光光度计(UV-Vis-NIR)，荧光分光光度计(2) Ultraviolet-visible-near-infrared spectrophotometer (UV-Vis-NIR), fluorescence spectrophotometer
紫外-可见-近红外吸收光谱，表示Pdots/Ce6lipid-Gd-DOTA纳米胶束合成的特征光谱。结果参照图2，(—)Pdots/Ce6lipid-Gd-DOTAmicelles的吸收光谱；(--)Ce6的吸收光谱；(-·-)表示Pdotslipidmicelles的吸收光谱；其中Pdots/Ce6lipid-Gd-DOTA纳米胶束在405nm及670nm具有比单独Pdotslipid不载有Ce6的纳米胶束更高的吸光度，表明Ce6已经成功的载入Pdots/Ce6lipid-Gd-DOTA内部。UV-Vis-NIR absorption spectrum, representing the characteristic spectrum of Pdots/Ce6lipid-Gd-DOTA nanomicelle synthesis. The results refer to Fig. 2, (—) the absorption spectrum of Pdots/Ce6lipid-Gd-DOTAmicelles; (--) the absorption spectrum of Ce6; (-·-) represents the absorption spectrum of Pdotslipidmicelles; Wherein Pdots/Ce6lipid-Gd-DOTA nanomicelle The absorbance at 405nm and 670nm is higher than that of Pdotslipid nanomicelles without Ce6, indicating that Ce6 has been successfully loaded into Pdots/Ce6lipid-Gd-DOTA.
(3)MRI磁共振成像和光声成像(3) MRI magnetic resonance imaging and photoacoustic imaging
MRI磁共振成像和光声成像图像，显示Pdots/Ce6lipid-Gd-DOTA纳米胶束具有磁共振成像和光声成像功能，结果参照图3，A图为Pdots/Ce6lipid-Gd-DOTA纳米胶束的T1磁共振图像，随着编号1,2，其浓度分别为0，0.1mM，其Pdots/Ce6lipid-Gd-DOTA纳米胶束的T1信号强度随浓度的增加而增强。B图为Pdots/Ce6lipid-Gd-DOTA纳米胶束的光声成像图，从左到右，Pdots的浓度依次为0,16ug/ml，表明Pdots/Ce6lipid-Gd-DOTA纳米胶束具有光声成像功能。MRI magnetic resonance imaging and photoacoustic imaging images, showing that Pdots/Ce6lipid-Gd-DOTA nanomicelles have the functions of magnetic resonance imaging and photoacoustic imaging, the results refer to Figure 3, A is the T1 of Pdots/Ce6lipid-Gd-DOTA nanomicelles In magnetic resonance images, along with No. 1 and No. 2, the concentrations are 0 and 0.1 mM respectively, and the T1 signal intensity of Pdots/Ce6lipid-Gd-DOTA nanomicelles increases with the increase of concentration. Figure B is the photoacoustic imaging of Pdots/Ce6lipid-Gd-DOTA nanomicelles. From left to right, the concentration of Pdots is 0, 16ug/ml, indicating that Pdots/Ce6lipid-Gd-DOTA nanomicelles have photoacoustic imaging Function.
(4)细胞杀伤能力；(4) cell killing ability;
利用不同浓度Pdots/Ce6lipid-Gd-DOTA纳米胶束，Ce6，Pdotslipid纳米胶束，如浓度为0.24,2.4μg/ml与肝癌细胞抚育，并在670nm近红外激光器照射5min后，利用细胞增殖与活性检测试剂盒(CCK8)(日本同仁化学研究所(上海))检测。结果参考图4，在670nm近红外激光的照射下，与单纯光动力或光热治疗相比，随着Pdots/Ce6lipid-Gd-DOTA纳米胶束的浓度增加，其细胞的存活率明显下降，说明光动力、光热治联合治疗能更为有效的抑制肿瘤细胞的增殖。Use different concentrations of Pdots/Ce6lipid-Gd-DOTA nanomicelles, Ce6, Pdotslipid nanomicelles, such as concentrations of 0.24, 2.4μg/ml to nurture liver cancer cells, and after 5 minutes of irradiation with a 670nm near-infrared laser, use cell proliferation and activity Detection kit (CCK8) (Japan Tongren Chemical Research Institute (Shanghai)) detection. The results refer to Figure 4. Under the irradiation of 670nm near-infrared laser, compared with pure photodynamic or photothermal therapy, the cell survival rate decreased significantly with the increase of the concentration of Pdots/Ce6lipid-Gd-DOTA nanomicelles, indicating that The combination of photodynamic therapy and photothermal therapy can more effectively inhibit the proliferation of tumor cells.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510429203.7ACN105012970B (en) | 2015-07-21 | 2015-07-21 | For the multifunctional nano micella and its application of early diagnosis of tumor and optical therapeutic |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510429203.7ACN105012970B (en) | 2015-07-21 | 2015-07-21 | For the multifunctional nano micella and its application of early diagnosis of tumor and optical therapeutic |
| Publication Number | Publication Date |
|---|---|
| CN105012970Atrue CN105012970A (en) | 2015-11-04 |
| CN105012970B CN105012970B (en) | 2018-05-29 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510429203.7AExpired - Fee RelatedCN105012970B (en) | 2015-07-21 | 2015-07-21 | For the multifunctional nano micella and its application of early diagnosis of tumor and optical therapeutic |
| Country | Link |
|---|---|
| CN (1) | CN105012970B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108653751A (en)* | 2017-03-29 | 2018-10-16 | 上海交通大学 | A kind of conjugated polymer nano-probe and its preparation method and application |
| CN108904818A (en)* | 2018-09-03 | 2018-11-30 | 国家纳米科学中心 | TTD core hybrid nanomaterial, preparation method and application |
| CN109010311A (en)* | 2018-09-28 | 2018-12-18 | 中国人民解放军陆军军医大学 | A kind of active oxygen responsiveness Moxifloxacin nanometer formulation and preparation method thereof |
| CN109420181A (en)* | 2017-08-23 | 2019-03-05 | 北京大学 | It is a kind of for tumour fluorescence imaging and photo-thermal/optical dynamic therapy multifunctional nanoparticle |
| CN109529059A (en)* | 2018-12-26 | 2019-03-29 | 中南大学 | A kind of fluorescence-magnetic resonance dual mode quantum dot and its preparation and application method |
| CN109602905A (en)* | 2018-12-11 | 2019-04-12 | 华中科技大学 | The application of micelles formed by amphiphilic molecules and Ag2S quantum dots as photosensitizers |
| CN110742588A (en)* | 2019-10-28 | 2020-02-04 | 四川大学华西医院 | Microwave thermoacoustic, photoacoustic and ultrasound three-modality intestinal tissue imaging method and system |
| CN110870920A (en)* | 2018-09-03 | 2020-03-10 | 国家纳米科学中心 | Hybrid nanoparticle, its preparation method and application |
| CN111035612A (en)* | 2019-12-31 | 2020-04-21 | 华中科技大学 | A reactive oxygen species responsive gel reservoir and its preparation method and application |
| CN111529704A (en)* | 2020-03-19 | 2020-08-14 | 中山大学附属第五医院 | Aggregation luminescence photosensitizer/antibacterial drug multifunctional nano micelle and preparation method and application thereof |
| CN112326580A (en)* | 2020-10-30 | 2021-02-05 | 深圳大学 | A material photodynamic performance testing system and its application |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103721271A (en)* | 2013-12-17 | 2014-04-16 | 福州市传染病医院 | Multifunctional nano probe for multimodal images and photothermal therapy of liver cancer and application of multifunctional nano probe |
| CN104364292A (en)* | 2012-04-13 | 2015-02-18 | 韦克森林大学 | Low band gap conjugated polymeric compositions and applications thereof |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104364292A (en)* | 2012-04-13 | 2015-02-18 | 韦克森林大学 | Low band gap conjugated polymeric compositions and applications thereof |
| CN103721271A (en)* | 2013-12-17 | 2014-04-16 | 福州市传染病医院 | Multifunctional nano probe for multimodal images and photothermal therapy of liver cancer and application of multifunctional nano probe |
| Title |
|---|
| KANYI PU ET AL.: "Semiconducting polymer nanoparticles as photoacoustic molecular imaging probes in living mice", 《NATURE NANOTECHNOLOGY》* |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108653751A (en)* | 2017-03-29 | 2018-10-16 | 上海交通大学 | A kind of conjugated polymer nano-probe and its preparation method and application |
| CN109420181A (en)* | 2017-08-23 | 2019-03-05 | 北京大学 | It is a kind of for tumour fluorescence imaging and photo-thermal/optical dynamic therapy multifunctional nanoparticle |
| CN108904818B (en)* | 2018-09-03 | 2022-02-18 | 国家纳米科学中心 | TTD (transthyretin) core hybrid nano-particles, and preparation method and application thereof |
| CN108904818A (en)* | 2018-09-03 | 2018-11-30 | 国家纳米科学中心 | TTD core hybrid nanomaterial, preparation method and application |
| CN110870920A (en)* | 2018-09-03 | 2020-03-10 | 国家纳米科学中心 | Hybrid nanoparticle, its preparation method and application |
| CN110870920B (en)* | 2018-09-03 | 2023-01-20 | 国家纳米科学中心 | Hybrid nanoparticles, method for the production thereof and use thereof |
| CN109010311A (en)* | 2018-09-28 | 2018-12-18 | 中国人民解放军陆军军医大学 | A kind of active oxygen responsiveness Moxifloxacin nanometer formulation and preparation method thereof |
| CN109602905A (en)* | 2018-12-11 | 2019-04-12 | 华中科技大学 | The application of micelles formed by amphiphilic molecules and Ag2S quantum dots as photosensitizers |
| CN109602905B (en)* | 2018-12-11 | 2022-03-29 | 华中科技大学 | Amphiphilic molecules with Ag2Application of micelle formed by S quantum dots as photosensitizer |
| CN109529059A (en)* | 2018-12-26 | 2019-03-29 | 中南大学 | A kind of fluorescence-magnetic resonance dual mode quantum dot and its preparation and application method |
| CN110742588A (en)* | 2019-10-28 | 2020-02-04 | 四川大学华西医院 | Microwave thermoacoustic, photoacoustic and ultrasound three-modality intestinal tissue imaging method and system |
| CN111035612A (en)* | 2019-12-31 | 2020-04-21 | 华中科技大学 | A reactive oxygen species responsive gel reservoir and its preparation method and application |
| CN111529704A (en)* | 2020-03-19 | 2020-08-14 | 中山大学附属第五医院 | Aggregation luminescence photosensitizer/antibacterial drug multifunctional nano micelle and preparation method and application thereof |
| CN112326580A (en)* | 2020-10-30 | 2021-02-05 | 深圳大学 | A material photodynamic performance testing system and its application |
| Publication number | Publication date |
|---|---|
| CN105012970B (en) | 2018-05-29 |
| Publication | Publication Date | Title |
|---|---|---|
| CN105012970B (en) | For the multifunctional nano micella and its application of early diagnosis of tumor and optical therapeutic | |
| Chen et al. | Noninvasively immunogenic sonodynamic therapy with manganese protoporphyrin liposomes against triple-negative breast cancer | |
| Hu et al. | Gadolinium-chelated conjugated polymer-based nanotheranostics for photoacoustic/magnetic resonance/NIR-II fluorescence imaging-guided cancer photothermal therapy | |
| Wang et al. | Fabrication of red blood cell-based multimodal theranostic probes for second near-infrared window fluorescence imaging-guided tumor surgery and photodynamic therapy | |
| Zhang et al. | Bioinspired multifunctional melanin-based nanoliposome for photoacoustic/magnetic resonance imaging-guided efficient photothermal ablation of cancer | |
| Liu et al. | Manganese (II) chelate functionalized copper sulfide nanoparticles for efficient magnetic resonance/photoacoustic dual-modal imaging guided photothermal therapy | |
| CN103721271B (en) | Multifunctional nano probe for multimodal images and photothermal therapy of liver cancer and application of multifunctional nano probe | |
| Jin et al. | Ultra-small iron-gallic acid coordination polymer nanoparticles for chelator-free labeling of 64 Cu and multimodal imaging-guided photothermal therapy | |
| Zhang et al. | Lipid micelles packaged with semiconducting polymer dots as simultaneous MRI/photoacoustic imaging and photodynamic/photothermal dual-modal therapeutic agents for liver cancer | |
| Wang et al. | Functionalized Cu 3 BiS 3 nanoparticles for dual-modal imaging and targeted photothermal/photodynamic therapy | |
| Liu et al. | Gd 3+-Ion-induced carbon-dots self-assembly aggregates loaded with a photosensitizer for enhanced fluorescence/MRI dual imaging and antitumor therapy | |
| Nafiujjaman et al. | Ternary graphene quantum dot–polydopamine–Mn 3 O 4 nanoparticles for optical imaging guided photodynamic therapy and T 1-weighted magnetic resonance imaging | |
| Liu et al. | Photosensitizer loaded PEG-MoS 2–Au hybrids for CT/NIRF imaging-guided stepwise photothermal and photodynamic therapy | |
| CN108434462B (en) | Multifunctional nano diagnosis and treatment agent with mesoporous polydopamine loaded carbonyl manganese and preparation method and application thereof | |
| Wang et al. | A triple-synergistic strategy for combinational photo/radiotherapy and multi-modality imaging based on hyaluronic acid-hybridized polyaniline-coated WS 2 nanodots | |
| Li et al. | A photosensitizer-conjugated magnetic iron oxide/gold hybrid nanoparticle as an activatable platform for photodynamic cancer therapy | |
| KR101739046B1 (en) | Nanoparticles for Diagnosis and Treatment of Tumor | |
| Wang et al. | Multifunctional mixed-metal nanoscale coordination polymers for triple-modality imaging-guided photodynamic therapy | |
| CN108888767B (en) | Nano composite material, preparation method and application thereof | |
| Zhang et al. | Gadolinium-chelate functionalized copper sulphide as a nanotheranostic agent for MR imaging and photothermal destruction of cancer cells | |
| Zhao et al. | High near-infrared absorbing Cu 5 FeS 4 nanoparticles for dual-modal imaging and photothermal therapy | |
| Zhang et al. | Theranostics for MRI‐guided therapy: recent developments | |
| CN107551279B (en) | Ultra-small protein composite nanoparticle with near-infrared photothermal effect and multi-modal imaging function, and preparation method and application thereof | |
| CN106512002B (en) | Multifunctional nano hybrid integrating CT imaging and phototherapy and preparation method thereof | |
| Chen et al. | Gadolinium–porphyrin based polymer nanotheranostics for fluorescence/magnetic resonance imaging guided photodynamic therapy |
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | Effective date of registration:20220228 Address after:430000 room 1203, 12th floor, complex building, taojialing 5 Industrial Park, taojialing community, Longyang street, Hanyang District, Wuhan City, Hubei Province Patentee after:Rongzhi Technology (Wuhan) Co.,Ltd. Address before:350025 No. 312 Xihong Road, Gulou District, Fuzhou City, Fujian Province Patentee before:FUZHOU HOSPITAL FOR INFECTIOUS DISEASE | |
| TR01 | Transfer of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee | Granted publication date:20180529 | |
| CF01 | Termination of patent right due to non-payment of annual fee |