WO2020132980A1

WO2020132980A1 - Bacterium-photothermal nanoparticle complex, preparation method therefor and use thereof

Info

Publication number: WO2020132980A1
Application number: PCT/CN2018/124022
Authority: WO
Inventors: 刘陈立; 蔡林涛; 黄建东; 臧中盛; 常志广; 夏霖; 曾正阳; 王伟
Original assignee: 深圳先进技术研究院
Priority date: 2018-12-26
Filing date: 2018-12-26
Publication date: 2020-07-02

Abstract

Disclosed are a bacterium-photothermal nanoparticle complex, a preparation method therefor and the use thereof, wherein same relate to the technical field of nanomedicine, the bacterium-photothermal nanoparticle complex comprises a photothermal nanoparticle and a bacterium, and the bacterium and the photothermal nanoparticle are connected by a chemical bond. The present invention improves the existing technical problem of the poor therapeutic effect of using bacteria to treat tumors, and also provides a bacterium-photothermal nanoparticle complex, wherein by chemically bonding a photothermal nanoparticle to a bacterium, the complex not only improves the safety and targeting of the bacterium in bodies, but can also realize the combination of bacterial therapy and photothermal therapy, and at the same time, same can also trace and image bacteria and tumors, and can effectively improve the effectiveness of tumor treatment.

Description

细菌-光热纳米颗粒复合物及制备方法和应用Bacteria-photothermal nanoparticle composite, preparation method and application

技术领域Technical field

本申请涉及纳米医学技术领域，尤其是涉及一种细菌-光热纳米颗粒复合物及制备方法和应用。The present application relates to the field of nanomedicine technology, and in particular to a bacteria-photothermal nanoparticle composite and its preparation method and application.

背景技术Background technique

癌症严重威胁着人类的健康，目前在临床上的治疗方法包括手术切除、放射性治疗、化学药物治疗以及近年来异军突起的细胞治疗和免疫治疗。尽管这些治疗方法能够延长病人的生存期，提高生存质量，但是依然存在一定的局限性。临床医生和科研人员始终在孜孜不倦地探索新的治疗方法为肿瘤治疗提供更多的选择方案。Cancer is a serious threat to human health. Current clinical treatment methods include surgical resection, radiotherapy, chemical drug treatment, and cell therapy and immunotherapy that have emerged in recent years. Although these treatments can prolong the patient's survival time and improve the quality of life, there are still certain limitations. Clinicians and researchers are always tirelessly exploring new treatments to provide more options for cancer treatment.

细菌在癌症治疗中的应用可以追溯到十九世纪晚期，但是使用细菌来治疗不可手术的肉瘤的审慎尝试也证实了该技术的固有危险，尽管采用细菌能够有效减少肿瘤和***，但是患者在治疗的9天死亡。因此，如何提高细菌治疗的安全性和有效性，成为人们关注的焦点。The use of bacteria in cancer treatment dates back to the late nineteenth century, but prudent attempts to use bacteria to treat inoperable sarcomas also confirmed the inherent dangers of the technology. Although the use of bacteria can effectively reduce tumors and lymph nodes, patients are treating 9 days of death. Therefore, how to improve the safety and effectiveness of bacterial treatment has become the focus of attention.

有鉴于此，特提出本申请。In view of this, this application is hereby submitted.

发明内容Summary of the invention

本申请的目的之一在于提供一种细菌-光热纳米颗粒复合物，以改善现有采用细菌***，治疗效果不佳的技术问题。One of the purposes of the present application is to provide a bacteria-photothermal nanoparticle composite to improve the existing technical problems of using bacteria to treat tumors and having a poor treatment effect.

本申请提供的细菌-光热纳米颗粒复合物，包括光热纳米颗粒和细菌，所述细菌与所述光热纳米颗粒通过化学键连接；The bacteria-photothermal nanoparticle composite provided by the present application includes photothermal nanoparticles and bacteria, and the bacteria and the photothermal nanoparticles are connected by a chemical bond;

优选地，所述细菌和所述光热纳米颗粒通过共价键连接。Preferably, the bacteria and the photothermal nanoparticles are connected by a covalent bond.

进一步地，所述细菌选自沙门氏菌、李斯特菌、大肠埃希氏菌和乳酸菌中的至少一种，优选为沙门氏菌，进一步优选为减毒沙门氏菌，更进一步优选为YB1；Further, the bacteria are selected from at least one of Salmonella, Listeria, Escherichia coli and lactic acid bacteria, preferably Salmonella, further preferably attenuated Salmonella, still more preferably YB1;

和/或，所述光热纳米颗粒包括光敏剂和纳米颗粒，所述光敏剂负载于所述纳米颗粒上，所述光敏剂选自ICG、PpIX和Ce6中的至少一种，优选为ICG；所述纳米颗粒选自磷脂聚合物纳米颗粒、PLGA、脂质体、金纳米笼、金纳米棒和介孔硅中的至少一种，优选为PLGA和磷脂聚合物纳米颗粒；And/or, the photothermal nanoparticles include photosensitizers and nanoparticles, the photosensitizers are supported on the nanoparticles, and the photosensitizers are selected from at least one of ICG, PpIX and Ce6, preferably ICG; The nanoparticles are selected from at least one of phospholipid polymer nanoparticles, PLGA, liposomes, gold nanocages, gold nanorods and mesoporous silicon, preferably PLGA and phospholipid polymer nanoparticles;

优选地，所述光热纳米颗粒的粒径为40-200nm，优选为50-100nm；Preferably, the particle size of the photothermal nanoparticles is 40-200nm, preferably 50-100nm;

优选地，所述光热纳米颗粒包载有药物，所述药物包括化疗药物和生物活性分子；Preferably, the photothermal nanoparticles are loaded with drugs, and the drugs include chemotherapy drugs and biologically active molecules;

优选地，所述化疗药物包括阿霉素类化疗药物、铂类化疗药、紫杉醇类化疗药物、IDO抑制剂、寡核苷酸DNA和脂多糖中的至少一种；Preferably, the chemotherapy drugs include at least one of adriamycin-based chemotherapy drugs, platinum-based chemotherapy drugs, paclitaxel-based chemotherapy drugs, IDO inhibitors, oligonucleotide DNA and lipopolysaccharide;

优选地，所述生物活性分子包括免疫检查点蛋白抑制剂、免疫激动剂、干扰素和白介素中的至少一种；Preferably, the biologically active molecules include at least one of immune checkpoint protein inhibitors, immune agonists, interferons and interleukins;

优选地，所述免疫激动剂包括PD-1单克隆抗体、PD-L1单克隆抗体、CTLA-4单克隆抗体、LAG-3单克隆抗体、TIM3单克隆抗体、TIGHT单克隆抗体和VISTA单克隆抗体中的至少一种；Preferably, the immune agonist includes PD-1 monoclonal antibody, PD-L1 monoclonal antibody, CTLA-4 monoclonal antibody, LAG-3 monoclonal antibody, TIM3 monoclonal antibody, TIGHT monoclonal antibody and VISTA monoclonal antibody At least one of the antibodies;

优选地，所述免疫激动剂包括4-1BB单克隆抗体和/或STING单克隆抗体。Preferably, the immune agonist comprises 4-1BB monoclonal antibody and/or STING monoclonal antibody.

进一步地，所述复合物包括YB1和光热纳米颗粒，所述YB1和所述光热纳米颗粒通过化学键连接，所述光热纳米颗粒包括ICG、PLGA和磷脂聚合物纳米颗粒；Further, the composite includes YB1 and photothermal nanoparticles, the YB1 and the photothermal nanoparticles are connected by a chemical bond, and the photothermal nanoparticles include ICG, PLGA, and phospholipid polymer nanoparticles;

优选地，所述ICG和所述PLGA包裹于所述磷脂聚合物纳米颗粒的内部，所述磷脂聚合物纳米颗粒和所述YB1通过化学键连接；Preferably, the ICG and the PLGA are wrapped inside the phospholipid polymer nanoparticles, and the phospholipid polymer nanoparticles and the YB1 are connected by a chemical bond;

进一步优选地，所述ICG负载于所述PLGA上。Further preferably, the ICG is loaded on the PLGA.

本申请的目的之二在于提供细菌-光热纳米颗粒复合物的制备方法，包括如下步骤：将细菌和光热纳米颗粒混合，使得细菌和光热纳米颗粒通过化学键连接，即得到细菌-光热纳米颗粒复合物。The second objective of the present application is to provide a method for preparing a bacteria-photothermal nanoparticle composite, including the following steps: mixing bacteria and photothermal nanoparticles so that the bacteria and photothermal nanoparticles are connected by a chemical bond to obtain bacteria-photothermal Nanoparticle composite.

进一步地，所述细菌-光热纳米颗粒复合物的制备方法包括如下步骤：Further, the preparation method of the bacteria-photothermal nanoparticle composite includes the following steps:

将细菌溶液、光热纳米颗粒和偶联剂混合均匀，使得细菌和光热纳米颗粒通过共价键连接，得到纳米颗粒复合物；Mix the bacterial solution, the photothermal nanoparticles and the coupling agent uniformly, so that the bacteria and the photothermal nanoparticles are connected by a covalent bond to obtain a nanoparticle composite;

优选地，所述细菌选自沙门氏菌、李斯特菌、大肠埃希氏菌和乳酸菌中的至少一种，优选为沙门氏菌，进一步优选为减毒沙门氏菌，更进一步优选为YB1；Preferably, the bacteria is selected from at least one of Salmonella, Listeria, Escherichia coli, and lactic acid bacteria, preferably Salmonella, further preferably attenuated Salmonella, even more preferably YB1;

优选地，所述光热纳米颗粒包括光敏剂和纳米颗粒，所述光敏剂负载于所述纳米颗粒上；所述光敏剂选自ICG、PpIX和Ce6中的至少一种，优选为ICG；所述纳米颗粒选自磷脂聚合物纳米颗粒、PLGA、脂质体、金纳米笼、金纳米棒和介孔硅中的至少一种，优选为磷脂聚合物纳米颗粒和PLGA；Preferably, the photothermal nanoparticles include photosensitizer and nanoparticles, and the photosensitizer is supported on the nanoparticles; the photosensitizer is selected from at least one of ICG, PpIX and Ce6, preferably ICG; The nanoparticles are selected from at least one of phospholipid polymer nanoparticles, PLGA, liposomes, gold nanocages, gold nanorods and mesoporous silicon, preferably phospholipid polymer nanoparticles and PLGA;

优选地，所述偶联剂为EDC和/或NHS。Preferably, the coupling agent is EDC and/or NHS.

所述光热纳米颗粒和所述细菌溶液的质量比为(0.5-1.5):10，优选为1:10；优选地，所述细菌溶液的菌落数(1-2)×10 ⁸cfu/mL，优选为1×10 ⁸cfu/mL。 The mass ratio of the photothermal nanoparticles to the bacterial solution is (0.5-1.5): 10, preferably 1:10; preferably, the number of colonies of the bacterial solution (1-2)×10 ⁸ cfu/mL , Preferably 1×10 ⁸ cfu/mL.

进一步地，所述光热纳米颗粒为ICG-PLGA-磷脂聚合物纳米颗粒，所述ICG-PLGA-磷脂聚合物纳米颗粒的制备方法包括如下步骤：Further, the photothermal nanoparticles are ICG-PLGA-phospholipid polymer nanoparticles, and the preparation method of the ICG-PLGA-phospholipid polymer nanoparticles includes the following steps:

(a)将大豆卵磷脂溶液、DSPE-PEG-COOH溶液和ICG溶液混合，得到混合溶液；(a) Mix the soybean lecithin solution, DSPE-PEG-COOH solution and ICG solution to obtain a mixed solution;

(b)将PLGA溶液在超声条件下加入上述混合溶液中，得到ICG-PLGA-磷脂聚合物纳米颗粒；其中，ICG-PLGA-磷脂聚合物纳米颗粒中，ICG和PLGA包裹于磷脂聚合物纳米颗粒的内部。(b) Add the PLGA solution to the above mixed solution under ultrasonic conditions to obtain ICG-PLGA-phospholipid polymer nanoparticles; wherein, in ICG-PLGA-phospholipid polymer nanoparticles, ICG and PLGA are wrapped in phospholipid polymer nanoparticles internal.

进一步地，PLGA、大豆卵磷脂、DSPE-PEG-COOH和ICG的质量比为(30-35):(2-4):(1-5):(10-17)，优选为33:3:2:15。Further, the mass ratio of PLGA, soybean lecithin, DSPE-PEG-COOH and ICG is (30-35):(2-4):(1-5):(10-17), preferably 33:3: 2:15.

本申请的目的之三在于提供上述细菌-光热纳米颗粒复合物在制备肿瘤治疗药物中的应用。The third object of the present application is to provide the application of the above-mentioned bacteria-photothermal nanoparticle complex in the preparation of tumor therapeutic drugs.

本申请提供的细菌-光热纳米颗粒复合物，通过将光热纳米颗粒化学键合于细菌上，不仅提高了细菌在体内的安全性和靶向性，而且能够实现细菌治疗和光热治疗的联合，同时还能够对细菌和肿瘤进行示踪和成像，从而有效提高肿瘤治疗的有效率。The bacteria-photothermal nanoparticle composite provided by this application not only improves the safety and targeting of bacteria in the body by chemically bonding the photothermal nanoparticles to the bacteria, but also enables the combination of bacterial therapy and photothermal therapy At the same time, it can also trace and image bacteria and tumors, thereby effectively improving the efficiency of tumor treatment.

附图说明BRIEF DESCRIPTION

图1为实施例3提供的INPs的SEM图；1 is a SEM image of INPs provided in Example 3;

图2为YB1的SEM图；Figure 2 is a SEM image of YB1;

图3为实施例8提供的YB1-INPs的SEM图；3 is an SEM image of YB1-INPs provided in Example 8;

图4为实施例3提供的INPs、YB1和实施例8提供的YB1-INPs的粒径分布图；4 is a particle size distribution diagram of INPs, YB1 provided in Example 3 and YB1-INPs provided in Example 8;

图5为实施例3提供的INPs的荧光图；5 is a fluorescence diagram of INPs provided in Example 3;

图6为YB1的荧光图；Figure 6 is the fluorescence diagram of YB1;

图7为实施例8提供的YB1-INPs的荧光图；7 is a fluorescence diagram of YB1-INPs provided in Example 8;

图8为试验例6中偶联在YB1上的INPs的数量与INPs浓度的关系图；8 is a graph showing the relationship between the number of INPs coupled to YB1 and the concentration of INPs in Test Example 6;

图9为试验例7中YB1培养24h后的活菌检测图；FIG. 9 is a test chart of live bacteria after 24 hours of YB1 culture in Test Example 7;

图10为试验例7中YB1-INPs培养24h后的活菌检测图；10 is a test chart of live bacteria after 24 hours of YB1-INPs culture in Test Example 7;

图11为试验例8中厌氧趋势性测试的装置结构示意图；11 is a schematic diagram of the structure of an anaerobic trend test device in Test Example 8;

图12为试验例8中在不同培养时间小室内YB1-INPs的数量柱状图；FIG. 12 is a histogram of the number of YB1-INPs in the test chamber 8 at different cultivation times;

图13为试验例8中培养40min后，在有氧和缺氧条件下小室内和小室外YB1-INPs比例的柱状图；FIG. 13 is a bar graph of the ratio of YB1-INPs in a small room and a small room under aerobic and anoxic conditions after 40 minutes of cultivation in Test Example 8;

图14a为试验例8中培养40min后，在有氧条件下，小室外YB1-INPs的荧光图；Fig. 14a is the fluorescence chart of YB1-INPs outside the cell under aerobic conditions after incubation for 40 minutes in Test Example 8;

图14b为试验例8中培养40min后，在缺氧条件下，小室外YB1-INPs的荧光图；Fig. 14b is the fluorescence graph of YB1-INPs outside the cell under hypoxic conditions after incubation for 40 minutes in Test Example 8;

图15为在试验例9中不同培养时间小室内YB1-INPs的数量柱状图；FIG. 15 is a histogram of the number of YB1-INPs in the cells of different cultivation times in Test Example 9;

图16为试验例9中培养40min后，在低氨基酸含量培养基和高氨基酸含量的培养基下小室内和小室外YB1-INPs比例的柱状图；16 is a bar graph of the ratio of YB1-INPs in a small room and a small room under a medium with a low amino acid content and a medium with a high amino acid content after 40 minutes of cultivation in Test Example 9;

图17a为试验例9中培养40min后，在低氨基酸培养基条件下，小室外YB1-INPs的荧光图；Fig. 17a is the fluorescence graph of YB1-INPs outside the cell under the condition of low amino acid medium after 40 minutes of cultivation in Test Example 9;

图17b为试验例9中培养40min后，在高氨基酸培养基条件下，小室外YB1-INPs的荧光图；Fig. 17b is the fluorescence graph of YB1-INPs outside the cell under the condition of high amino acid medium after 40 minutes of cultivation in Test Example 9;

图18为试验例10中3只小鼠荧光成像图；Fig. 18 is a fluorescence imaging diagram of three mice in Test Example 10;

图19为试验例10中注射72h后，3只小鼠肿瘤部位的荧光强度柱状图；19 is a histogram of the fluorescence intensity of the tumor site of three mice 72 hours after injection in Test Example 10;

图20为试验例10中注射72h后，3只小鼠肿瘤部位的免疫组化图；FIG. 20 is an immunohistochemical diagram of tumor sites of three mice 72 hours after injection in Test Example 10;

图21为试验例10中注射72h后，第二只小鼠和第三只小鼠心、肝、脾、肺、肾脏和肿瘤组织的YB1含量图；21 is a graph of YB1 content of heart, liver, spleen, lung, kidney and tumor tissues of the second and third mice 72 hours after injection in Test Example 10;

图22为试验例10中注射72h后，第二只小鼠和第三只小鼠肿瘤组织中YB1数量的柱状图；22 is a histogram of the number of YB1 in the tumor tissues of the second mouse and the third mouse 72 hours after injection in Test Example 10;

图23为试验例11中注射72h后，三只小鼠部位温度和近红外激光照射时间的关系图；FIG. 23 is a graph showing the relationship between the temperature of three mice and the irradiation time of near-infrared laser light after 72 hours of injection in Test Example 11;

图24为试验例12中4只小鼠在不同时间的肿瘤部位状态图；FIG. 24 is a diagram of the tumor sites of four mice in Test Example 12 at different times;

图25为试验例13中4组小鼠肿瘤平均体积和时间的关系图；25 is a graph showing the relationship between the average tumor volume and time of the four groups of mice in Test Example 13;

图26为试验例14中4组小鼠生存率和时间的关系图；FIG. 26 is a graph showing the relationship between survival rate and time of four groups of mice in Test Example 14;

图27为试验例15中2只小鼠心、肝、脾、肺和肾脏组织染色图。FIG. 27 is a staining chart of heart, liver, spleen, lung and kidney tissues of two mice in Test Example 15. FIG.

具体实施方式detailed description

下面将对本申请的技术方案进行清楚且完整地描述，显然，所描述的实施例是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，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，都属于本申请保护的范围。The technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, but not all of them. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without making creative efforts fall within the protection scope of the present application.

根据本申请的一个方面，本申请提供了一种细菌-光热纳米颗粒复合物，包括光热纳米颗粒和细菌，所述细菌与所述光热纳米颗粒通过化学键连接。According to one aspect of the present application, the present application provides a bacteria-photothermal nanoparticle composite, including photothermal nanoparticles and bacteria, and the bacteria and the photothermal nanoparticles are connected by a chemical bond.

在本申请中，细菌-光热纳米颗粒复合物中的“-”含义为“和”。In this application, the "-" in the bacteria-photothermal nanoparticle composite means "and".

在本申请中，化学键连接包括但不限于离子连接、共价键连接和金属键连接。In the present application, chemical bonding includes, but is not limited to, ionic bonding, covalent bonding, and metal bonding.

典型但非限制性的实现化学键连接的方式有：细菌和光热纳米颗粒均修饰有功能基团，细菌和光热纳米颗粒通过各自功能基团实现化学键连接。Typical but non-limiting ways of achieving chemical bonding are: bacteria and photothermal nanoparticles are modified with functional groups, and bacteria and photothermal nanoparticles are chemically bonded through their respective functional groups.

在本申请的一种优选实施方式中，细菌-光热纳米颗粒复合物中，细菌和光热纳米颗粒通过共价键连接。通过将细菌和光热纳米颗粒通过共价键连接，使得细菌和光热纳米颗粒之间的连接更为牢固，从而使得细菌-光热纳米颗粒复合物在体内的稳定性更佳。In a preferred embodiment of the present application, in the bacteria-photothermal nanoparticle composite, the bacteria and the photothermal nanoparticles are connected by a covalent bond. By connecting the bacteria and the photothermal nanoparticles through a covalent bond, the connection between the bacteria and the photothermal nanoparticles is made stronger, so that the stability of the bacteria-photothermal nanoparticle composite in the body is better.

在本申请的一种优选实施方式中，细菌包括但不限于沙门氏菌(Salmonella Typhimurium)、李斯特菌(Listeria monocytogenes)、大肠埃希氏菌(E.coli)和乳酸菌(Lactic acid bacteria)。In a preferred embodiment of the present application, the bacteria include, but are not limited to, Salmonella (Typhimurium), Listeria monocytogenes, E. coli, and Lactic acid bacteria.

在本申请的一种优选实施方式中，细菌为沙门氏菌，优选为减毒沙门氏菌，更优选为YB1。减毒沙门氏菌制备抗肿瘤药物，安全性更好，而YB1在有氧环境中无法生长会裂解死亡，在肿瘤的厌氧区域环境下才能够生长，所以YB1具有实体瘤肿瘤具有靶向性，对正常组织器官的毒性小，安全性好。在本申请的一种优选实施方式中，光热纳米颗粒包括光敏剂和纳米颗粒，所述光敏剂负载于所述纳米颗粒上，光敏剂包括ICG(吲哚菁绿)、PpIX(原卟啉)和Ce6中的一种或几种，如光敏剂为ICG和PpIX、PpIX、Ce6或ICG。In a preferred embodiment of the present application, the bacteria is Salmonella, preferably attenuated Salmonella, more preferably YB1. Anti-tumor drugs prepared by attenuated Salmonella have better safety, and YB1 can not grow in an aerobic environment and will lyse and die. It can only grow in the anaerobic area of the tumor. Therefore, YB1 has solid tumors. Normal tissues and organs are less toxic and safe. In a preferred embodiment of the present application, the photothermal nanoparticles include photosensitizers and nanoparticles, the photosensitizers are supported on the nanoparticles, and the photosensitizers include ICG (indocyanine green), PpIX (protoporphyrin ) And one or more of Ce6, for example, the photosensitizer is ICG and PpIX, PpIX, Ce6 or ICG.

在本申请的优选实施方式中，通过将光敏剂负载于纳米颗粒上，以提高光敏性的稳定性，使其能够实现纳米颗粒的示踪功能和光热治疗功能。In a preferred embodiment of the present application, by loading the photosensitizer on the nanoparticles, the stability of photosensitivity is improved, so that it can realize the tracking function and photothermal therapy function of the nanoparticles.

在本申请的优选实施方式中，光敏剂为ICG。ICG常作为血管造影剂使用，其安全性高。在本申请的优选实施方式中，将ICG用于制备细菌-光热纳米颗粒复合物，不仅安全性好，而且具有较高的光热转化效率，从而能够有效提高细菌-光热纳米颗粒复合物的治疗效果。In a preferred embodiment of the present application, the photosensitizer is ICG. ICG is often used as an angiography agent, and its safety is high. In a preferred embodiment of the present application, ICG is used to prepare a bacteria-photothermal nanoparticle composite, which not only has good safety, but also has a high photothermal conversion efficiency, which can effectively improve the bacteria-photothermal nanoparticle composite Treatment effect.

在本申请的一种优选实施方式中，纳米颗粒选自PLGA(聚乳酸-羟基乙酸共聚物)、脂质体、金纳米笼、金纳米棒和介孔硅中的至少一种，如纳米颗粒为PLGA和脂质体、金纳米笼和脂质体或脂质体和介孔硅。In a preferred embodiment of the present application, the nanoparticles are selected from at least one of PLGA (polylactic acid-glycolic acid copolymer), liposomes, gold nanocages, gold nanorods, and mesoporous silicon, such as nanoparticles For PLGA and liposomes, gold nanocages and liposomes or liposomes and mesoporous silicon.

在本申请的进一步优选实施方式中，纳米颗粒为PLGA和磷脂聚合物纳米颗粒。PLGA和磷脂聚合物纳米颗粒能够在体内生物降解，而金纳米笼、金纳米棒和介孔硅作为纳米颗粒不易在体内代谢，因此，选用PLGA和磷脂聚合物纳米颗粒作为负载细菌的载体时，更易于在体内进行生物降解，其作为载体负载细菌时进入体内***时，其生物相容性更佳。In a further preferred embodiment of the present application, the nanoparticles are PLGA and phospholipid polymer nanoparticles. PLGA and phospholipid polymer nanoparticles can be biodegraded in vivo, while gold nanocage, gold nanorods and mesoporous silicon as nanoparticles are not easy to metabolize in vivo. Therefore, when PLGA and phospholipid polymer nanoparticles are used as carriers for bacteria, It is easier to biodegrade in the body, and when it is used as a carrier to load bacteria into the body to treat tumors, its biocompatibility is better.

在本申请的一种优选实施方式中，光热纳米颗粒的粒径为40-200nm，优选为50-100nm。在本申请的优选实施方式中，光热纳米颗粒的典型但非限制性的粒径如为40、50、60、70、80、90、100、110、120、130、140、150、180或200nm。In a preferred embodiment of the present application, the particle diameter of the photothermal nanoparticles is 40-200 nm, preferably 50-100 nm. In a preferred embodiment of the present application, the typical but non-limiting particle sizes of the photothermal nanoparticles are 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 180 or 200nm.

通过将光热纳米颗粒的粒径控制为40-200nm，以提高光热纳米颗粒的靶向性，当光热纳米颗粒的粒径小于40nm时，容易发生团聚，不易于与细菌进行化学键链接，而且肿瘤被动靶向富集效果差(EPR 效应)。如果光热纳米颗粒的粒径超过200nm，则容易被体内的免疫细菌捕获，无法到达肿瘤部位。尤其是当光热纳米颗粒的粒径为50-100nm时，其作为细菌载体在体内的靶向性更佳。By controlling the particle diameter of the photothermal nanoparticles to 40-200nm, to improve the targeting of the photothermal nanoparticles, when the particle diameter of the photothermal nanoparticles is less than 40nm, it is easy to agglomerate, and it is not easy to chemically link with bacteria. Moreover, the effect of passive targeted enrichment of tumors is poor (EPR effect). If the particle diameter of the photothermal nanoparticles exceeds 200 nm, it is easily captured by immune bacteria in the body and cannot reach the tumor site. Especially when the particle size of the photothermal nanoparticles is 50-100 nm, its targeting as a bacterial carrier in the body is better.

在本申请的一种优选实施方式中，光热纳米颗粒包载有药物，药物包括但不限于化疗药物和具有生物活性的分子。In a preferred embodiment of the present application, the photothermal nanoparticles are loaded with drugs. The drugs include but are not limited to chemotherapeutic drugs and molecules with biological activity.

在本申请的优选实施方式中，通过采用光热纳米颗粒包载药物，使得细菌-光热纳米颗粒能够同时实现细菌治疗、光热治疗和药物治疗，从而进一步提高肿瘤的治疗效果。In a preferred embodiment of the present application, the use of photothermal nanoparticles to encapsulate drugs enables bacterial-photothermal nanoparticles to simultaneously realize bacterial therapy, photothermal therapy, and drug therapy, thereby further improving the therapeutic effect of tumors.

在本申请的一种优选实施方式中，所述药物包括化疗药物和生物活性分子；In a preferred embodiment of the present application, the drugs include chemotherapeutic drugs and biologically active molecules;

在本申请的进一步优选实施方式中，所述化疗药物包括但不限于阿霉素类化疗药物，铂类化疗药，紫杉醇类化疗药物、IDO抑制剂，寡核苷酸DNA和脂多糖中的一种或几种。In a further preferred embodiment of the present application, the chemotherapeutic drugs include but are not limited to one of doxorubicin chemotherapeutics, platinum chemotherapeutics, paclitaxel chemotherapeutics, IDO inhibitors, oligonucleotide DNA and lipopolysaccharide Kind or several.

在本申请的进一步优选实施方式中，所述生物活性分子包括但不限于免疫检查点蛋白抑制剂，如PD-1/PD-L1单克隆抗体，CTLA-4单克隆抗体，LAG-3单克隆抗体，TIM3单克隆抗体，TIGHT单克隆抗体，VISTA单克隆抗体，免疫激动剂，如4-1BB单克隆抗体，STING单克隆抗体；具有治疗效果的干扰素和白介素等。In a further preferred embodiment of the present application, the biologically active molecules include, but are not limited to, immune checkpoint protein inhibitors, such as PD-1/PD-L1 monoclonal antibody, CTLA-4 monoclonal antibody, LAG-3 monoclonal antibody Antibody, TIM3 monoclonal antibody, TIGHT monoclonal antibody, VISTA monoclonal antibody, immune agonist, such as 4-1BB monoclonal antibody, STING monoclonal antibody; interferon and interleukin with therapeutic effect.

在本申请的一种优选实施方式中，细菌-光热纳米颗粒复合物包括YB1和光热纳米颗粒，所述YB1和所述光热纳米颗粒通过化学键连接。通过选用YB1与光热纳米颗粒通过化学键连接后***，其安全性更佳。In a preferred embodiment of the present application, the bacterial-photothermal nanoparticle composite includes YB1 and photothermal nanoparticles, and the YB1 and the photothermal nanoparticles are connected by a chemical bond. By selecting YB1 and photothermal nanoparticles to be connected by chemical bonds to treat tumors, its safety is better.

在本申请的一种优选实施方式中，YB1上设有功能基团，光热纳米颗粒上也设有功能基团，YB1和光热纳米颗粒通过各自的功能基团实现化学键连接。In a preferred embodiment of the present application, YB1 is provided with functional groups, and the photothermal nanoparticles are also provided with functional groups, and YB1 and the photothermal nanoparticles are chemically bonded through their respective functional groups.

在本申请的典型但非限制性的实施方式中，YB1上设置有氨基，光热纳米颗粒上设置有羧基，YB1和光热纳米颗粒通过氨基和羧基之间的形成酰胺基连接。In a typical but non-limiting embodiment of the present application, an amino group is provided on YB1, and a carboxyl group is provided on the photothermal nanoparticles, and YB1 and the photothermal nanoparticles are connected by forming an amide group between the amino group and the carboxyl group.

在本申请的一种优选实施方式中，光热纳米颗粒包括ICG、PLGA和脂质体，其中，ICG和所述PLGA包裹于脂质体的内部，所述脂质体和所述YB1通过化学键连接。通过采用脂质体包裹ICG和PLGA，以提高ICG的稳定性，避免其在体内发生淬灭，影响示踪功能和光热治疗功能。In a preferred embodiment of the present application, the photothermal nanoparticles include ICG, PLGA and liposome, wherein ICG and the PLGA are encapsulated inside the liposome, and the liposome and the YB1 are chemically bonded connection. Encapsulation of ICG and PLGA by liposomes improves the stability of ICG, avoids its quenching in the body, and affects the tracer function and photothermal therapy function.

在本申请的进一步优选实施方式中，ICG负载于PLGA上。通过采用PLGA负载ICG，以进一步提高ICG的稳定性，从而进一步提高YB1-ICG-PLGA-磷脂聚合物纳米颗粒在体内的示踪稳定性和光热治疗效果。In a further preferred embodiment of the present application, the ICG is loaded on PLGA. By using PLGA to load ICG, the stability of ICG is further improved, thereby further improving the tracking stability and photothermal treatment effect of YB1-ICG-PLGA-phospholipid polymer nanoparticles in vivo.

根据本申请的第二个方面，本申请提供了一种细菌-光热纳米颗粒复合物的制备方法，包括如下步骤：According to the second aspect of the present application, the present application provides a method for preparing a bacterial-photothermal nanoparticle composite, including the following steps:

将细菌和光热纳米颗粒混合，使得细菌和光热纳米颗粒通过化学键连接，即得到细菌-光热纳米颗粒复合物。The bacteria and the photothermal nanoparticles are mixed, so that the bacteria and the photothermal nanoparticles are connected by a chemical bond to obtain the bacteria-photothermal nanoparticle composite.

在本申请的典型但非限制性的实施方式中，细菌和光热纳米颗粒上均修饰有功能基团，细菌和光热纳米颗粒通过各自功能基团之间的相互作用，实现化学键连接。In a typical but non-limiting embodiment of the present application, functional groups are modified on both the bacteria and the photothermal nanoparticles, and the bacteria and the photothermal nanoparticles achieve chemical bond connection through the interaction between the respective functional groups.

在本申请中，化学键连接包括但不限于离子连接、共价键连接和金属键连接。本申请提供的细菌-光热纳米颗粒的制备方法，工艺简单，操作方便，能够进行批量生产，降低制备成本。In this application, chemical bonding includes but is not limited to ionic bonding, covalent bonding, and metal bonding. The preparation method of the bacterial-photothermal nanoparticles provided by the present application has a simple process and convenient operation, can be mass-produced, and reduces the preparation cost.

在本申请的一种优选实施方式中，细菌-光热纳米颗粒复合物的制备方法包括如下步骤：In a preferred embodiment of the present application, the preparation method of the bacteria-photothermal nanoparticle composite includes the following steps:

将细菌溶液、光热纳米颗粒和偶联剂混合均匀，使得细菌和光热纳米颗粒通过共价键连接，得到纳米颗粒复合物。The bacterial solution, the photothermal nanoparticles and the coupling agent are uniformly mixed, so that the bacteria and the photothermal nanoparticles are connected by a covalent bond to obtain a nanoparticle composite.

在本申请的优选实施方式中，通过加入偶联剂，以活化细菌上的功能基团和光热纳米颗粒上的功能基团，从而使得细菌和光热纳米颗粒通过共价键连接。In a preferred embodiment of the present application, a coupling agent is added to activate the functional group on the bacteria and the functional group on the photothermal nanoparticles, so that the bacteria and the photothermal nanoparticles are connected by a covalent bond.

在本申请的一种优选方式中，细菌的选择和光热纳米颗粒的选择如前所述，在此不再赘述。In a preferred mode of the present application, the selection of bacteria and the selection of photothermal nanoparticles are as described above, and will not be repeated here.

在本申请的一种优选实施方式中，偶联剂为EDC和/或NHS，其中，EDC为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，NHS为N-羟基琥珀酸亚胺。In a preferred embodiment of the present application, the coupling agent is EDC and/or NHS, wherein EDC is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, NHS It is N-hydroxysuccinic imine.

在本申请的一种优选实施方式中，复合物由YB1溶液、ICG-PLGA-磷脂聚合物纳米颗粒和偶联剂制备而成，其制备方法包括如下步骤：将YB1溶液、ICG-PLGA-磷脂聚合物纳米颗粒和偶联剂混合均匀，得到YB1-ICG-PLGA-磷脂聚合物纳米颗粒复合物。In a preferred embodiment of the present application, the complex is prepared from a YB1 solution, ICG-PLGA-phospholipid polymer nanoparticles and a coupling agent. The preparation method includes the following steps: the YB1 solution, ICG-PLGA-phospholipid The polymer nanoparticles and the coupling agent are uniformly mixed to obtain a YB1-ICG-PLGA-phospholipid polymer nanoparticle composite.

在本申请的优选实施方式中，YB1修饰有氨基，ICG-PLGA-磷脂聚合物纳米颗粒带有羧基，通过加入偶联剂，以使得ICG-PLGA-磷脂聚合物纳米颗粒表面的羧基和YB1上的氨基发生反应，形成酰胺键，从而得到YB1和ICG-PLGA-磷脂聚合物纳米颗粒通过酰胺键连接的YB1-ICG-PLGA-磷脂聚合物纳米颗粒复合物。In a preferred embodiment of the present application, YB1 is modified with an amino group, and ICG-PLGA-phospholipid polymer nanoparticles have carboxyl groups. By adding a coupling agent, the carboxyl groups on the surface of ICG-PLGA-phospholipid polymer nanoparticles and YB1 The amino group of the reaction occurs to form an amide bond, thereby obtaining a YB1-ICG-PLGA-phospholipid polymer nanoparticle composite of YB1 and ICG-PLGA-phospholipid polymer nanoparticles connected by an amide bond.

在本申请的一种优选实施方式中，光热纳米颗粒和细菌溶液的质量比为(0.5-1.5):10，以提高YB1-ICG-PLGA-磷脂聚合物纳米颗粒复合物中光热纳米颗粒的负载率，从而进一步提高肿瘤的光热治疗效果。In a preferred embodiment of the present application, the mass ratio of the photothermal nanoparticles to the bacterial solution is (0.5-1.5): 10, to improve the photothermal nanoparticles in the YB1-ICG-PLGA-phospholipid polymer nanoparticle composite The loading rate, thereby further improving the photothermal treatment effect of the tumor.

典型但非限制性地，光热纳米颗粒和细菌溶液的质量比如为0.5:10、0.6:10、0.7:10、0.8:10、0.9:10、1:10、1.1:10、1.2:10、1.3:10、1.4:10或1.5:10。Typical but non-limiting, the masses of photothermal nanoparticles and bacterial solutions are, for example, 0.5:10, 0.6:10, 0.7:10, 0.8:10, 0.9:10, 1:10, 1.1:10, 1.2:10, 1.3:10, 1.4:10 or 1.5:10.

在本申请的一种优选实施方式中，细菌溶液的菌落数为(1-2)×10 ⁸cfu/mL。 In a preferred embodiment of the present application, the bacterial solution has a colony number of (1-2)×10 ⁸ cfu/mL.

典型但非限制性地，细菌溶液的菌落数为1×10 ⁸cfu/mL、1.1×10 ⁸cfu/mL、1.2×10 ⁸cfu/mL、1.3×10 ⁸cfu/mL、1.4×10 ⁸cfu/mL、1.5×10 ⁸cfu/mL、1.6×10 ⁸cfu/mL、1.7×10 ⁸cfu/mL、1.8×10 ⁸cfu/mL、1.9×10 ⁸cfu/mL或2×10 ⁸cfu/mL。 Typical but non-limiting, the bacterial solution colony count is 1×10 ⁸ cfu/mL, 1.1×10 ⁸ cfu/mL, 1.2×10 ⁸ cfu/mL, 1.3×10 ⁸ cfu/mL, 1.4×10 ⁸ cfu /mL, 1.5×10 ⁸ cfu/mL, 1.6×10 ⁸ cfu/mL, 1.7×10 ⁸ cfu/mL, 1.8×10 ⁸ cfu/mL, 1.9×10 ⁸ cfu/mL, or 2×10 ⁸ cfu/mL .

在本申请的一种优选实施方式中，光热纳米颗粒为ICG-PLGA-磷脂聚合物纳米颗粒，其制备方法包括如下步骤：In a preferred embodiment of the present application, the photothermal nanoparticles are ICG-PLGA-phospholipid polymer nanoparticles, and the preparation method includes the following steps:

(a)将大豆卵磷脂溶液、DSPE-PEG-COOH溶液和ICG溶液混合均匀，得到混合溶液；(a) Mix the soybean lecithin solution, DSPE-PEG-COOH solution and ICG solution uniformly to obtain a mixed solution;

在本申请的一种优选实施方式中，PLGA、大豆卵磷脂、DSPE-PEG-COOH和ICG的质量比为(30-35):(2-4):(1-5):(10-17)，优选为33:3:2:15。In a preferred embodiment of the present application, the mass ratio of PLGA, soybean lecithin, DSPE-PEG-COOH and ICG is (30-35):(2-4):(1-5):(10-17 ), preferably 33:3:2:15.

在本申请的优选实施方式中，通过磷脂聚合物纳米颗粒包裹ICG和PLGA，以进一步提高ICG在体内的稳定性，避免其发生淬灭，从而进一步提高YB1-ICG-PLGA-磷脂聚合物纳米颗粒在体内的示踪稳定性和光热治疗功能。In a preferred embodiment of the present application, ICG and PLGA are encapsulated by phospholipid polymer nanoparticles to further improve the stability of ICG in vivo and avoid quenching, thereby further improving YB1-ICG-PLGA-phospholipid polymer nanoparticles In vivo tracking stability and photothermal therapy function.

通过控制PLGA、大豆卵磷脂、DSPE-PEG-COOH和ICG的质量比，以使得ICG和PLGA纳米颗粒被完全包覆于磷脂聚合物纳米颗粒中。By controlling the mass ratio of PLGA, soybean lecithin, DSPE-PEG-COOH and ICG, the ICG and PLGA nanoparticles are completely coated in the phospholipid polymer nanoparticles.

在本申请的一种优选实施方式中，在步骤(a)中，ICG溶液的溶剂为乙醇和水的混合溶液，其中，乙醇和水体积比为(3-6):(94-97)，优选为4:96。通过选用乙醇和水的混合溶液作为ICG的溶剂，以提高ICG的溶解性。In a preferred embodiment of the present application, in step (a), the solvent of the ICG solution is a mixed solution of ethanol and water, wherein the volume ratio of ethanol and water is (3-6): (94-97), It is preferably 4:96. By using a mixed solution of ethanol and water as the ICG solvent, the solubility of ICG is improved.

在本申请的优选实施方式中，ICG溶液的溶剂中，乙醇和水的典型但非限制性的体积比如为3:97、4:96、5:95或6:94。In a preferred embodiment of the present application, in the solvent of the ICG solution, typical but non-limiting volumes of ethanol and water are, for example, 3:97, 4:96, 5:95, or 6:94.

在本申请的一种优选实施方式中，PLGA溶液的溶剂为乙腈。In a preferred embodiment of the present application, the solvent of the PLGA solution is acetonitrile.

在本申请的一种优选实施方式中，在步骤(b)中，大豆卵磷脂溶液的溶剂为氯仿和甲醇的混合溶液，其中，氯仿和甲醇的体积比为(8-10):1。通过选用氯仿和甲醇的混合溶液作为大豆卵磷脂的溶剂，以提高大豆卵磷脂的溶解性。In a preferred embodiment of the present application, in step (b), the solvent of the soybean lecithin solution is a mixed solution of chloroform and methanol, wherein the volume ratio of chloroform to methanol is (8-10):1. By using the mixed solution of chloroform and methanol as the solvent of soybean lecithin, the solubility of soybean lecithin can be improved.

在本申请的优选实施方式中，大豆卵磷脂溶液的溶剂中，氯仿和甲醇的典型但非限制性的体积比为8:1、9:1或10:1。在本申请的一种优选实施方式中，DSPE-PEG-COOH溶液的溶剂为乙醇和水的混合溶液，其中，乙醇和水的体积比为(2-8):(92-98)。通过选用乙醇和水的混合溶液作为DSPE-PEG-COOH的溶剂，以提高DSPE-PEG-COOH的溶解性。In a preferred embodiment of the present application, in the solvent of the soybean lecithin solution, a typical but non-limiting volume ratio of chloroform and methanol is 8:1, 9:1 or 10:1. In a preferred embodiment of the present application, the solvent of the DSPE-PEG-COOH solution is a mixed solution of ethanol and water, wherein the volume ratio of ethanol and water is (2-8):(92-98). By using the mixed solution of ethanol and water as the solvent of DSPE-PEG-COOH, the solubility of DSPE-PEG-COOH is improved.

在本申请优选实施方式中，DSPE-PEG-COOH溶液的溶剂中，乙醇和水的典型但非限制性的体积比如为2:98、3:97、4:96、5:95、6:94、7:93或8:92。在本申请的一种优选实施方式中，YB1溶液的溶剂为PBS溶液。In a preferred embodiment of the present application, in the solvent of the DSPE-PEG-COOH solution, typical but non-limiting volumes of ethanol and water are, for example, 2:98, 3:97, 4:96, 5:95, 6:94 , 7:93 or 8:92. In a preferred embodiment of the present application, the solvent of the YB1 solution is PBS solution.

在本申请的一种优选实施方式中，在步骤(a)中，超声的功率为35-45W，频率为15-25Hz，时间为2-5min。通过控制超声的功率为35-45W，频率为15-25Hz，时间为2-5min，以使得ICG和PLGA充分接触，提高PLGA上负载的ICG的数量，尤其是当超声的功率为38-40W，频率为18-22Hz，时间为2-4min，ICG和PLGA接触得更加充分，PLGA上负载的ICG的数量更多。In a preferred embodiment of the present application, in step (a), the ultrasonic power is 35-45 W, the frequency is 15-25 Hz, and the time is 2-5 min. By controlling the ultrasonic power to 35-45W, frequency 15-25Hz, time 2-5min, to make ICG and PLGA fully contact, increase the number of ICG loaded on PLGA, especially when the ultrasonic power is 38-40W, The frequency is 18-22Hz, the time is 2-4min, ICG and PLGA contact more fully, the number of ICG loaded on PLGA is more.

在本申请的优选实施方式中，超声的典型但非限制性的功率如为35、36、37、38、39、40、41、42、 43、44或45W；典型但非限制性的频率如为15、16、17、18、19、20、21、22、23、24或25Hz；典型但非限制性的时间如为2、3、4或5min。根据本申请的第三个方面，本申请提供了细菌-光热纳米颗粒复合物在制备肿瘤治疗药物中的应用。In a preferred embodiment of the present application, the typical but non-limiting power of ultrasound is 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45W; the typical but non-limiting frequency is such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 Hz; typical but non-limiting times are 2, 3, 4 or 5 min. According to the third aspect of the present application, the present application provides the application of the bacterial-photothermal nanoparticle complex in the preparation of tumor therapeutic drugs.

下面结合实施例对本申请提供的技术方案做进一步的描述。The technical solution provided by the present application will be further described below in conjunction with the embodiments.

实施例1Example 1

本实施例提供了一种ICG-PLGA-磷脂聚合物纳米颗粒(以下简称INPs)，其按照如下步骤制备而成：This embodiment provides an ICG-PLGA-phospholipid polymer nanoparticles (hereinafter referred to as INPs), which are prepared according to the following steps:

(1)将PLGA溶解于乙腈中，得到浓度为2mg/mL的PLGA溶液；将ICG溶解于乙醇和水的混合溶液(乙醇浓度为4％)中，得到浓度为0.75mg/mL的ICG溶液；大豆卵磷脂溶解于氯仿和甲醇的混合溶液(氯仿和甲醇体积比为9:1)中，得到大豆卵磷脂溶液；将DSPE-PEG-COOH溶解于乙醇和水的混合溶液(乙醇浓度为4％)中，得到DSPE-PEG-COOH溶液；(1) Dissolve PLGA in acetonitrile to obtain a PLGA solution with a concentration of 2 mg/mL; dissolve ICG in a mixed solution of ethanol and water (ethanol concentration is 4%) to obtain an ICG solution with a concentration of 0.75 mg/mL; Soy lecithin was dissolved in a mixed solution of chloroform and methanol (chloroform and methanol volume ratio of 9:1) to obtain a soybean lecithin solution; DSPE-PEG-COOH was dissolved in a mixed solution of ethanol and water (ethanol concentration is 4%) ), get DSPE-PEG-COOH solution;

(2)在超声条件下，将大豆卵磷脂溶液、DSPE-PEG-COOH溶液和ICG溶液混合，得到混合溶液；(2) Mix the soybean lecithin solution, DSPE-PEG-COOH solution and ICG solution under ultrasonic conditions to obtain a mixed solution;

(3)将PLGA溶液在超声条件下加入上述混合溶液中，得到ICG-PLGA-磷脂聚合物纳米颗粒；(3) Add the PLGA solution to the above mixed solution under ultrasonic conditions to obtain ICG-PLGA-phospholipid polymer nanoparticles;

其中，PLGA、大豆卵磷脂、DSPE-PEG-COOH和ICG的质量比为30:3:5:17。Among them, the mass ratio of PLGA, soybean lecithin, DSPE-PEG-COOH and ICG is 30:3:5:17.

实施例2Example 2

本实施例提供了一种INPs，其制备方法与实施例1的不同之处在于，在步骤(3)中，PLGA、大豆卵磷脂、DSPE-PEG-COOH和ICG-PLGA纳米颗粒的质量比为35:4:3:10。This example provides an INPs. The preparation method differs from Example 1 in that in step (3), the mass ratio of PLGA, soybean lecithin, DSPE-PEG-COOH, and ICG-PLGA nanoparticles is 35:4:3:10.

实施例3Example 3

本实施例提供了一种INPs，其制备方法与实施例1的不同之处在于，在步骤(3)中，大豆卵磷脂、DSPE-PEG-COOH和ICG-PLGA纳米颗粒的质量比为33:3:2:15。This example provides an INPs. The preparation method is different from Example 1 in that in step (3), the mass ratio of soybean lecithin, DSPE-PEG-COOH, and ICG-PLGA nanoparticles is 33: 3:2:15.

实施例4Example 4

本实施例提供了一种INPs，其制备方法与实施例1的不同之处在于，在步骤(3)中，PLGA、大豆卵磷脂、DSPE-PEG-COOH和ICG-PLGA纳米颗粒的质量比为20:3:2:15。This example provides an INPs. The preparation method differs from Example 1 in that in step (3), the mass ratio of PLGA, soybean lecithin, DSPE-PEG-COOH, and ICG-PLGA nanoparticles is 20:3:2:15.

实施例5Example 5

本实施例提供了一种INPs，其制备方法与实施例1的不同之处在于，在步骤(3)中，PLGA、大豆卵磷脂、DSPE-PEG-COOH和ICG-PLGA纳米颗粒的质量比为20:3:2:5。This example provides an INPs. The preparation method differs from Example 1 in that in step (3), the mass ratio of PLGA, soybean lecithin, DSPE-PEG-COOH, and ICG-PLGA nanoparticles is 20:3:2:5.

试验例1Test Example 1

将实施例1-5提供的INPs通过透射电镜和马尔文颗粒表征分析仪进行检测，结果显示，实施例1-3提供的INPs的粒径均一性更好，粒径分布在80-120nm，且光热纳米颗粒全部被包覆于脂质体中，而实施例4-5提供的INPs的粒径均一性稍差，粒径分布在50-200nm，且有部分光热纳米颗粒未被完全包覆，这说明在制备INPs时，PLGA、大豆卵磷脂、DSPE-PEG-COOH和ICG-PLGA纳米颗粒的质量比为(30-35):(2-4):(1-5):(10-17)时，制备得到的INPs的粒径分布更均一，光热纳米颗粒包覆更完全。The INPs provided in Examples 1-5 were detected by transmission electron microscopy and Malvern particle characterization analyzer. The results showed that the INPs provided in Examples 1-3 had better particle size uniformity and the particle size distribution was 80-120 nm, and The photothermal nanoparticles are all encapsulated in liposomes, while the particle size uniformity of the INPs provided in Example 4-5 is slightly worse, the particle size distribution is between 50-200 nm, and some photothermal nanoparticles are not completely encapsulated This shows that the mass ratio of PLGA, soybean lecithin, DSPE-PEG-COOH and ICG-PLGA nanoparticles is (30-35):(2-4):(1-5):(10 -17), the particle size distribution of the prepared INPs is more uniform, and the photothermal nanoparticles are coated more completely.

实施例6Example 6

本实施例提供了一种YB1-ICG-PLGA-磷脂聚合物纳米颗粒复合物(以下简称YB1-INPs)，其由实施例3提供的ICG-PLGA-磷脂聚合物纳米颗粒和YB1制备而成，具体包括如下步骤：This example provides a YB1-ICG-PLGA-phospholipid polymer nanoparticle composite (hereinafter referred to as YB1-INPs), which is prepared from the ICG-PLGA-phospholipid polymer nanoparticles and YB1 provided in Example 3. It includes the following steps:

将ICG-PLGA-磷脂聚合物纳米颗粒、EDC和YB1溶液混合均匀，得到YB1-INPs，其中，YB1溶液的菌落数为1×10 ⁸cfu/mL；ICG-PLGA-磷脂聚合物纳米颗粒和YB1溶液的质量比为1:10。 Mix the ICG-PLGA-phospholipid polymer nanoparticles, EDC and YB1 solution uniformly to obtain YB1-INPs, where the number of colonies in the YB1 solution is 1×10 ⁸ cfu/mL; ICG-PLGA-phospholipid polymer nanoparticles and YB1 The mass ratio of the solution is 1:10.

实施例7Example 7

本实施例提供了一种YB1-INPs，其制备方法与实施例6的不同之处在于，ICG-PLGA-磷脂聚合物纳米颗粒和YB1溶液的质量比为0.5:10。This example provides a YB1-INPs. The preparation method is different from that in Example 6 in that the mass ratio of the ICG-PLGA-phospholipid polymer nanoparticles and the YB1 solution is 0.5:10.

实施例8Example 8

本实施例提供了一种YB1-INPS，其制备方法与实施例6的不同之处在于，ICG-PLGA-磷脂聚合物纳米颗粒和YB1溶液的质量比为1.5:10。This example provides a YB1-INPS. The preparation method differs from Example 6 in that the mass ratio of ICG-PLGA-phospholipid polymer nanoparticles and YB1 solution is 1.5:10.

实施例9Example 9

本实施例提供了一种YB1-INPs，其制备方法与实施例6的不同之处在于，ICG-PLGA-磷脂聚合物纳米颗粒和YB1溶液的质量比为0.2:10。This example provides a YB1-INPs. The preparation method differs from Example 6 in that the mass ratio of the ICG-PLGA-phospholipid polymer nanoparticles and the YB1 solution is 0.2:10.

实施例10Example 10

本实施例提供了一种YB1-INP _S，其制备方法与实施例6的不同之处在于，ICG-PLGA-磷脂聚合物纳米颗粒和YB1溶液的质量比为5:10。 This example provides a YB1-INP _S. The preparation method differs from Example 6 in that the mass ratio of the ICG-PLGA-phospholipid polymer nanoparticles and the YB1 solution is 5:10.

试验例2Test Example 2

将实施例6-10提供的YB1-INPs利用激光共聚焦显微镜观察荧光，结果显示实施例6-8提供的YB1-INPs的荧光强度大于实施例9-10提供的YB1-INPs，这说明实施例6-8提供的YB1-INPs中细菌上负载的ICG-PLGA-磷脂聚合物纳米颗粒高于实施例9-10，这说明在制备YB1-INPs时，ICG-PLGA-磷脂聚合物纳米颗粒和YB1溶液的质量比为(0.5-1.5):10，且YB1溶液的菌落数为(1-2)×10 ⁸cfu/mL时，其制备得到的YB1-INPs负载的ICG-PLGA-磷脂聚合物纳米颗粒更高，其对肿瘤的治疗效果更好。 The YB1-INPs provided in Example 6-10 were observed with a laser confocal microscope. The results showed that the fluorescence intensity of YB1-INPs provided in Example 6-8 was greater than that of YB1-INPs provided in Example 9-10, which illustrates the example 6-8 The ICG-PLGA-phospholipid polymer nanoparticles loaded on the bacteria in YB1-INPs provided are higher than those in Examples 9-10, which shows that when preparing YB1-INPs, ICG-PLGA-phospholipid polymer nanoparticles and YB1 When the mass ratio of the solution is (0.5-1.5):10, and the number of colonies of the YB1 solution is (1-2)×10 ⁸ cfu/mL, the ICG-PLGA-phospholipid polymer nanoparticles supported on the YB1-INPs prepared by the preparation The higher the particle size, the better the treatment effect on the tumor.

试验例3Test Example 3

将实施例3提供的INPs、YB1和实施例8提供的YB1-INPs采用扫描电镜进行观测，其结果如图1-3所示，其中图1为实施例3提供的INPs的SEM图；图2为YB1的SEM图；图3为实施例8提供的YB1-INPs的SEM图；从图1可以看出，INPs具有明显的核壳结构，且其粒径为100nm左右；从图2可以看出，YB1的粒径为1μm左右；从图3可以看出，YB1-INPs的粒径为1μm左右，且YB1表面有凸起状纳米颗粒，这说明INPs成功负载于YB1表面。The INPs and YB1 provided in Example 3 and the YB1-INPs provided in Example 8 were observed by scanning electron microscopy. The results are shown in Figures 1-3. Figure 1 is the SEM image of the INPs provided in Example 3; Figure 2 It is the SEM image of YB1; Figure 3 is the SEM image of YB1-INPs provided in Example 8; as can be seen from Figure 1, INPs have a clear core-shell structure, and its particle size is about 100nm; can be seen from Figure 2 The particle size of YB1 is about 1 μm; as can be seen from Figure 3, the particle size of YB1-INPs is about 1 μm, and there are convex nanoparticles on the surface of YB1, which shows that INPs are successfully loaded on the surface of YB1.

试验例4Test Example 4

将实施例3提供的INPs、YB1和实施例8提供的YB1-INPs分别采用马尔文颗粒表征分析仪进行分析，结果如图4所示，从图4可以看出，实施例3提供的INPs粒径分布在50-100nm，YB1和实施例8提供的YB1-INPs的粒径分布在800-1200nm。The INPs and YB1 provided in Example 3 and the YB1-INPs provided in Example 8 were analyzed using a Malvern particle characterization analyzer. The results are shown in FIG. 4. As can be seen from FIG. 4, the INPs particles provided in Example 3 The diameter distribution is 50-100 nm, and the particle size distribution of YB1 and YB1-INPs provided in Example 8 is 800-1200 nm.

试验例5Test Example 5

将实施例3提供的INPs、YB1和实施例8提供的YB1-INPs分别采用激光共聚焦显微镜观察荧光，结果如图5-7所示，其中，图5为实施例3提供的INPs的荧光图；图6为YB1的荧光图；图7为实施例8提供的YB1-INPs的荧光图；从图5-7可以看出，INPs具有荧光示踪功能，YB1没有荧光示踪功能，YB1-INPs具有荧光示踪功能。The INPs, YB1 provided in Example 3, and YB1-INPs provided in Example 8 were used to observe fluorescence using a laser confocal microscope, respectively. The results are shown in Figures 5-7, where Figure 5 is the fluorescence image of the INPs provided in Example 3. Figure 6 is the fluorescence diagram of YB1; Figure 7 is the fluorescence diagram of YB1-INPs provided in Example 8; as can be seen from Figures 5-7, INPs have a fluorescent tracking function, YB1 does not have a fluorescent tracking function, YB1-INPs With fluorescent tracking function.

试验例6Test Example 6

将实施例3提供的INPs分别配置成浓度为0、50μg/mL、100μg/mL和150μg/mL，其分别和10 ⁸cfu/g的YB1在平板上进行孵育，孵育2h后，偶联在YB1上的INPs的数量与INPs浓度的关系如图8所示，从图8可以看出，当INPs的浓度为0-100μg/mL时，随着INPs浓度的增高，偶联在YB1上的INPs数量增加，但是当INPS的浓度为100-150μg/mL时，偶联在YB1上的INPs数量没有明显变化。 After INPs 3 embodiment are arranged to provide a concentration of 0,50μg / mL, 100μg / mL and 150μg / mL, respectively, and 10 ⁸ cfu / g of YB1 were incubated on the plate, incubated for 2h, the coupling YB1 The relationship between the number of INPs and the concentration of INPs is shown in Figure 8. As can be seen from Figure 8, when the concentration of INPs is 0-100μg/mL, as the concentration of INPs increases, the number of INPs coupled to YB1 Increased, but when the concentration of INPS was 100-150 μg/mL, the number of INPs coupled to YB1 did not change significantly.

另外，在图8中插图分别为浓度为0、50μg/mL、100μg/mL和150μg/mL和10 ⁸cfu/mL的YB1在平板上孵育2h后的近红外照相后的图像，从插图也可以看出，当INPs的浓度为0-100μg/mL时，随着INPs浓度的增高，偶联在YB1上的INPs数量增加，但是当INP _S的浓度为100-150μg/mL时，偶联在YB1上的INPs数量没有明显变化。 In addition, the insets in FIG. 8 are the images after near-infrared photography of YB1 with concentrations of 0, 50 μg/mL, 100 μg/mL, 150 μg/mL, and 10 ⁸ cfu/mL incubated on the plate for 2 hours. It can be seen that when the concentration of INPs is 0-100 μg/mL, as the concentration of INPs increases, the number of INPs coupled to YB1 increases, but when the concentration of INP _S is 100-150 μg/mL, the coupling at YB1 There was no significant change in the number of INPs.

试验例7Test Example 7

取YB1和实施例8提供的YB1-INPs分别培养24h，其中YB1和YB1-INPs具有相同活菌数量，结果如图9-10所示，图9为YB1培养24h后的活菌检测图；图10为YB1-INPs培养24h后的活菌检测图；在图9和图10中，上部为细菌原液点板，下部为细菌原液稀释10倍后的点板，从图9和图10可以看出，YB1和实施例8提供的YB1-INPs分别培养24h后，其活菌数量相当，这说明在YB1上偶联INPs后，不会影响YB1的生长。YB1 and YB1-INPs provided in Example 8 were cultured for 24 hours, of which YB1 and YB1-INPs had the same number of live bacteria. The results are shown in Figures 9-10. Figure 9 is the live bacteria detection chart after YB1 cultured for 24 hours; 10 is the live bacteria detection chart after YB1-INPs culture for 24h; in Figure 9 and Figure 10, the upper part is the bacterial stock spot plate, and the lower part is the bacterial spot diluted 10 times, as can be seen from Figure 9 and Figure 10 After YB1-INPs provided in Example 8 and YB1-INPs were cultured for 24 hours, the number of viable bacteria was equivalent. This shows that the coupling of INPs on YB1 will not affect the growth of YB1.

试验例8Test Example 8

将实施例8提供的YB1-INPs进行厌氧趋势性测试，测试步骤如下：The YB1-INPs provided in Example 8 were subjected to an anaerobic trend test. The test steps are as follows:

(1)在6孔板中铺设小鼠乳腺癌细胞系4T1；(1) Lay the mouse breast cancer cell line 4T1 in a 6-well plate;

(2)在6孔板中***侵袭小室，如图11所示，侵袭小室的底部设置有碳酸酯膜，侵袭小室中加入一定数量的YB1-INPs，通过碳酸酯膜将YB1-INPs和6孔板下层的肿瘤细胞分隔；(2) Insert the invasion cell into the 6-well plate. As shown in Figure 11, the bottom of the invasion cell is provided with a carbonate film. A certain amount of YB1-INPs is added to the invasion cell. The YB1-INPs and 6 holes are inserted through the carbonate film. Separation of tumor cells in the lower layer of the plate;

(3)分别在有氧和缺氧条件下，将YB1-INPs和肿瘤细菌共培养40min，然后利用流式细胞仪对小室内和小室外的YB1-INPs进行计数统计。(3) Under aerobic and hypoxic conditions, YB1-INPs and tumor bacteria were co-cultured for 40 min, and then flow cytometry was used to count YB1-INPs in the indoor and outdoor chambers.

图12为在不同培养时间小室内YB1-INPs的数量柱状图，其中Normoxia代表有氧条件，Hypoxia代表缺氧条件，从图12可以看出，在缺氧条件下小室中YB1-INPs的数量显著低于有氧条件下小室中YB1-INPs的数量，这说明在缺氧条件下，YB1-INPs更容易穿透碳酸酯膜，进入小室外，靶向肿瘤细胞。Figure 12 is a histogram of the number of YB1-INPs in the chamber at different cultivation times, where Normoxia represents aerobic conditions and Hypoxia represents anoxic conditions. As can be seen from Figure 12, the number of YB1-INPs in the chamber is significant under hypoxic conditions It is lower than the number of YB1-INPs in the chamber under aerobic conditions, which means that under hypoxic conditions, YB1-INPs can more easily penetrate the carbonate membrane and enter the chamber to target tumor cells.

图13为培养40min后，在有氧和缺氧条件下小室内和小室外YB1-INPs比例的柱状图，其中，Supernatant代表小室内，Bottom chamber代表小室外，Normoxia代表有氧条件，Hypoxia代表缺氧条件；从图12可以看出，在缺氧条件下，小室外的YB1-INPs的比例为52％，小室内的YB1-INPs的比例为48％，而在有氧条件下，小室外的YB1-INPs的比例为20％，小室内的YB1-INPs的比例为80％，这也说明在缺氧条件下，YB1-INPs更容易穿透碳酸酯膜，进入小室外，靶向肿瘤细胞。Figure 13 is a histogram of the proportion of YB1-INPs in the small room and the small room under aerobic and anoxic conditions after 40 minutes of cultivation, where Supernatant stands for the small room, Bottom chamber stands for the small room, Normoxia stands for the aerobic conditions, and Hypoxia stands for the lack of Oxygen conditions; as can be seen from Figure 12, under hypoxic conditions, the proportion of YB1-INPs in the small room is 52%, the proportion of YB1-INPs in the small room is 48%, and under aerobic conditions, the The proportion of YB1-INPs is 20%, and the proportion of YB1-INPs in the cell is 80%, which also shows that under hypoxic conditions, YB1-INPs can more easily penetrate the carbonate membrane, enter the cell, and target tumor cells.

图14a和图14b分别为培养40min后，在有氧和缺氧条件下，小室外YB1-INPs的荧光图，其中，Normoxia代表有氧条件，Hypoxia代表缺氧条件；从图14a和图14b可以看出，在缺氧条件下小室外的荧光强度限于高于有氧条件下小室外的荧光强度，这说明在缺氧条件下，YB1-INPs更容易穿透碳酸酯膜，进入小室外，靶向肿瘤细胞。Figures 14a and 14b are the fluorescence graphs of YB1-INPs outside the cell after aerobic and anoxic conditions after 40 minutes of cultivation, where Normoxia represents aerobic conditions and Hypoxia represents anoxic conditions; from Figures 14a and 14b It can be seen that the fluorescence intensity outside the cell under anoxic conditions is limited to that higher than that under aerobic conditions, which means that under anoxic conditions, YB1-INPs can more easily penetrate the carbonate membrane and enter the cell outside. To tumor cells.

试验例9Test Example 9

(2)在6孔板中***侵袭小室，其结构同图11，侵袭小室的底部设置有碳酸酯膜，侵袭小室中加入一定数量的YB1-INPs，通过碳酸酯膜将YB1-INPs和6孔板下层的肿瘤细胞分隔；(2) Insert the invasion cell into the 6-well plate. Its structure is the same as that in Figure 11. The bottom of the invasion cell is provided with a carbonate film. A certain amount of YB1-INPs is added to the invasion cell. The YB1-INPs and 6 holes are added through the carbonate film. Separation of tumor cells in the lower layer of the plate;

(3)分别将6孔板中的培养基更换为低氨基酸含量培养基和高氨基酸含量的培养基，将YB1-INPs和肿瘤细菌共培养40min，然后利用流式细胞仪对小室内和小室外的YB1-INPs进行计数统计。(3) Replace the medium in the 6-well plate with a medium with low amino acid content and a medium with high amino acid content, incubate YB1-INPs and tumor bacteria for 40 min, and then use a flow cytometer to test the indoor and outdoor YB1-INPs for counting statistics.

图15为在不同培养时间小室内YB1-INPs的数量柱状图，其中Nutrition代表高氨基酸含量的培养基，PBS代表低氨基酸含量培养基，从图15可以看出，在使用高氨基酸含量的培养基时，小室中YB1-INPs的数量显著高于使用低氨基酸含量的培养基下小室中YB1-INPs的数量，这说明在低氨基酸含量培养基培养时，YB1-INPs更容易穿透碳酸酯膜，进入小室外，靶向肿瘤细胞。Figure 15 is a histogram of the number of YB1-INPs in a small chamber at different cultivation times, where Nutrition represents a medium with a high amino acid content and PBS represents a medium with a low amino acid content. As can be seen from FIG. 15, when a medium with a high amino acid content is used At that time, the number of YB1-INPs in the chamber was significantly higher than the number of YB1-INPs in the chamber with low amino acid content medium, which means that when cultured in low amino acid content medium, YB1-INPs are more likely to penetrate the carbonate membrane, Enter the cell and target tumor cells.

图16为培养40min后，在低氨基酸含量培养基和高氨基酸含量的培养基下小室内和小室外YB1-INPs比例的柱状图，其中，Supernatant代表小室内，Bottom chamber代表小室外，Nutrition代表高氨基酸含量的培养基，PBS代表低氨基酸含量培养基；从图16可以看出，在高氨基酸含量的培养基培养下，小室外的YB1-INPs的比例为52％，小室内的YB1-INPs的比例为48％左右，而在低氨基酸含量的培养基培养下，小室外的YB1-INPs的比例为80％，小室内的YB1-INPs的比例为20％，这也说明在高氨基酸含量的培养基培养条件下，YB1-INPs更容易穿透碳酸酯膜，进入小室外，靶向肿瘤细胞。16 is a histogram of the ratio of YB1-INPs in the small room and the small room under the medium with low amino acid content and the medium with high amino acid content after 40 minutes of cultivation, where Supernatant represents the small room, Bottom chamber represents the small room, and Nutrition represents the high Amino acid content medium, PBS stands for low amino acid content medium; as can be seen from Figure 16, under the high amino acid content medium culture, the proportion of YB1-INPs outside the cell is 52%, and the YB1-INPs inside the cell The ratio is about 48%, and in the culture medium with low amino acid content, the proportion of YB1-INPs in the small room is 80%, and the proportion of YB1-INPs in the small room is 20%, which also shows that in the cultivation of high amino acid content Under basal culture conditions, YB1-INPs can more easily penetrate the carbonate membrane, enter the cell, and target tumor cells.

图17a和图17b为培养40min后，在低氨基酸含量培养基和高氨基酸含量的培养基条件下，小室外YB1-INPs的荧光图，其中Nutrition代表高氨基酸含量的培养基，PBS代表低氨基酸含量培养基；从图17a和17b可以看出，在高氨基酸含量的培养基条件下小室外的荧光强度限于高于低氨基酸含量的培养基条件下小室外的荧光强度，这说明在高氨基酸含量的培养基条件下，YB1-INPs更容易穿透碳酸酯膜，进入小室外，靶向肿瘤细胞。Figures 17a and 17b are fluorescence diagrams of YB1-INPs outside the cell after low-amino acid medium and high amino acid content after 40 minutes of cultivation, where Nutrition represents high amino acid content and PBS represents low amino acid content Medium; as can be seen from Figures 17a and 17b, the fluorescence intensity in the cell outdoors under the condition of medium with high amino acid content is limited to the fluorescence intensity in the cell outdoors under the condition of medium with low amino acid content, which shows that Under medium conditions, YB1-INPs can more easily penetrate the carbonate membrane, enter the cell, and target tumor cells.

试验例10Test Example 10

取具有相同肿瘤，且肿瘤体积相同的同种类小鼠3只，第一只小鼠注射INPs溶液，第二只小鼠注射YB1-INPs溶液，第三只小鼠注射YB1-INPs溶液，其中，三种注射液的荧光强度相同，注射量相同，且第三只小鼠在注射12h，采用近红外激光照射5分钟(808纳米，1.18W/cm ²)。 Three mice of the same type with the same tumor and the same tumor volume were taken. The first mouse was injected with INPs solution, the second mouse was injected with YB1-INPs solution, and the third mouse was injected with YB1-INPs solution. The three injections had the same fluorescence intensity and the same injection volume, and the third mouse was injected with a near-infrared laser for 5 minutes (808 nm, 1.18 W/cm ² ) for 12 h.

分别在3只小鼠注射后1h、12h、24h、48h和72h对小鼠进行荧光成像，且在72h后，将三只小鼠处死，并分别将肿瘤组织取出进行荧光成像，如图18所示，其中，INPs代表第一只小鼠，YB1-INPs代表第二只小鼠，YB1-INPs(+)代表第三只小鼠。Three mice were fluorescently imaged at 1h, 12h, 24h, 48h and 72h after injection, and after 72h, the three mice were sacrificed and tumor tissues were taken out for fluorescence imaging, as shown in Figure 18 Shown, where INPs represent the first mouse, YB1-INPs represent the second mouse, and YB1-INPs (+) represent the third mouse.

从图18可以看出，第一只小鼠注射INPs后，INPs进入小鼠体内，大部分被免疫***阻挡，在72h后仅有极少数INPs在肿瘤部位聚集；第二只小鼠注射YB1-INPs后，部分YB1-INPs被免疫***阻挡，但是有部分YB1-INPs聚集在肿瘤部位；这三只小鼠注射YB1-INPs后，小部分YB1-INPs被免疫***阻挡，大部分YB1-INPs聚集在肿瘤部位；这说明采用YB1-INPs更易于靶向于肿瘤部位，尤其是在注射YB1-INPs后，辅以近红外激光照射更能够提高YB1-INPs在肿瘤部位的聚集率，提高治疗效果。As can be seen from Figure 18, after the first mouse was injected with INPs, the INPs entered the mouse body, most of which were blocked by the immune system, and only a few INPs accumulated at the tumor site after 72h; the second mouse was injected with YB1- After INPs, some YB1-INPs were blocked by the immune system, but some YB1-INPs gathered at the tumor site; after the injection of YB1-INPs in these three mice, a small part of YB1-INPs were blocked by the immune system, and most of the YB1-INPs gathered At the tumor site; this shows that YB1-INPs are easier to target at the tumor site, especially after injection of YB1-INPs, supplemented with near-infrared laser irradiation can improve the aggregation rate of YB1-INPs at the tumor site and improve the therapeutic effect.

图19为注射后72h，3只小鼠肿瘤部位的荧光强度柱状图，其中，INPs代表第一只小鼠，YB1-INPs代表第二只小鼠，YB1-INPs(+)代表第三只小鼠；从图19可以看出，第一只小鼠肿瘤部位的荧光强度很弱，这说明仅有极少数INPs在肿瘤组织聚集；第二只小鼠肿瘤部位的荧光强度明显高于第一只小鼠，这说明第二只小鼠肿瘤部位聚集的YB1-INPs的数量显著高于第一只；第三只小鼠肿瘤部位的荧光强度明显高于第二只小鼠，这说明在进行细菌治疗时，辅以近红外激光照射更能够提高YB1-INPs在肿瘤部位的聚集率，从而提高治疗效果。Fig. 19 is a histogram of the fluorescence intensity of the tumor site of 3 mice 72h after injection, in which INPs represent the first mouse, YB1-INPs represent the second mouse, and YB1-INPs (+) represent the third small Mouse; as can be seen from Figure 19, the fluorescence intensity of the tumor site of the first mouse is very weak, indicating that only a few INPs accumulate in the tumor tissue; the fluorescence intensity of the tumor site of the second mouse is significantly higher than that of the first Mice, which means that the number of YB1-INPs accumulated in the tumor site of the second mouse is significantly higher than that of the first mouse; the fluorescence intensity of the tumor site of the third mouse is significantly higher than that of the second mouse, which means that the bacteria During treatment, supplemented with near-infrared laser irradiation can increase the aggregation rate of YB1-INPs at the tumor site, thereby improving the treatment effect.

图20为注射后72h，3只小鼠肿瘤部位的免疫组化图，其中箭头表示YB1所在位置，从图20可以看出，第一只小鼠肿瘤部位未发现YB1，第二只小鼠肿瘤部位存在YB1，但其YB1数量明显低于第三只小鼠肿瘤部位的YB1数量，这说明YB1-INPs比INPs更易于在肿瘤部位聚集，尤其辅以红外激光照射时，YB1-INPs在肿瘤部位的聚集率更高，治疗效果更好。Figure 20 is the immunohistochemical image of the tumor site of 3 mice 72h after injection, where the arrow indicates the location of YB1, as can be seen from Figure 20, the tumor site of the first mouse was not found YB1, the tumor of the second mouse YB1 is present in the site, but the number of YB1 is significantly lower than that of the third mouse tumor site, which shows that YB1-INPs are more likely to accumulate in the tumor site than INPs, especially when supplemented by infrared laser irradiation, YB1-INPs are in the tumor site The aggregation rate is higher and the treatment effect is better.

在注射72h后，分别将第二只小鼠和第三只小鼠处死，并取出心、肝、脾、肺、肾脏和肿瘤组织进行YB1含量检测，结果如图21所示，其中，YB1-INPs代表第二只小鼠，YB1-INPs+Laser代表第三只小鼠，从图21可以看出，第三只小鼠中聚集在肿瘤部位的YB1的数量显著高于第二只小鼠，辅以红外激光照射时，YB1-INPs在肿瘤部位的聚集率更高，治疗效果更好。72h after injection, the second mouse and the third mouse were sacrificed, and the heart, liver, spleen, lung, kidney and tumor tissues were taken out for YB1 content detection. The results are shown in Figure 21, where YB1- INPs represents the second mouse, and YB1-INPs+Laser represents the third mouse. As can be seen from FIG. 21, the number of YB1 accumulated in the tumor site of the third mouse is significantly higher than that of the second mouse. When supplemented with infrared laser irradiation, the accumulation rate of YB1-INPs at the tumor site is higher, and the treatment effect is better.

图22为注射72h后，第二只小鼠和第三只小鼠肿瘤组织的YB1数量的柱状图，其中，YB1-INPs代表第二只小鼠，YB1-INPs+Laser代表第三只小鼠，从图22可以看出，第三只小鼠中聚集在肿瘤部位的YB1的数量显著高于第二只小鼠，辅以红外激光照射时，YB1-INPs在肿瘤部位的聚集率更高，治疗效果更好。Fig. 22 is a histogram of the number of YB1 in the tumor tissues of the second and third mice 72 hours after injection, where YB1-INPs represents the second mouse and YB1-INPs+Laser represents the third mouse It can be seen from Fig. 22 that the number of YB1 accumulated in the tumor site in the third mouse was significantly higher than that in the second mouse. When supplemented with infrared laser irradiation, the aggregation rate of YB1-INPs in the tumor site was higher. The treatment effect is better.

试验例11Test Example 11

取具有相同肿瘤，且肿瘤体积相同的同种类小鼠3只，第一只小鼠注射PBS溶液，第二只小鼠注射INPs溶液，第三只小鼠注射YB1-INPs溶液，其中三只小鼠的注射量相同，且三只小鼠在注射12h和72h，分别采用近红外激光照射450s，(808纳米，1.18W/cm ²)。 Three mice of the same type with the same tumor and the same tumor volume were taken. The first mouse was injected with PBS solution, the second mouse was injected with INPs solution, and the third mouse was injected with YB1-INPs solution, three of which were small The injection volume of the mice was the same, and the three mice were irradiated with near-infrared laser for 450 s (808 nm, 1.18 W/cm ² ) at 12 h and 72 h respectively.

注射72h后，分别监测不同照射时间时三只小鼠肿瘤部位的温度，绘制不同照射时间和肿瘤部位温度的关系图，如图23所示，其中PBS(++)代表第一只小鼠，INPs(++)代表第二只小鼠，YB1-INPs(++)代表第三只小鼠。从图23可以看出，随着照射时间的延长，第一只小鼠肿瘤部位的温度并无明显变化，第二只小鼠肿瘤部位的温度随着照射时间的延长，稍有升高，但是最高温度仍在40℃以下，第三只小鼠随着照射时间的延长，肿瘤部位的温度显著提高，最高温度达到65℃，这说明采用YB1-INPs进行肿瘤治疗，并辅以近红外激光照射时，肿瘤部位的温度最高，肿瘤的治疗效果更好，采用INPs进行肿瘤治疗，并辅以近红外激光照射，肿瘤部位温度稍有升高，肿瘤治疗效果显著低于YB1-INPs，而PBS溶液则不会对肿瘤部位的温度产生明显影响。72 hours after injection, the temperature of the tumor site of the three mice at different irradiation times were monitored, and the relationship between the different irradiation time and the tumor site temperature was plotted, as shown in Figure 23, where PBS(++) represents the first mouse, INPs(++) represents the second mouse, and YB1-INPs(++) represents the third mouse. As can be seen from Figure 23, with the extension of the irradiation time, the temperature of the tumor site of the first mouse did not change significantly, and the temperature of the tumor site of the second mouse increased slightly with the extension of the exposure time, but The maximum temperature is still below 40°C. The temperature of the tumor area of the third mouse increased significantly with the extension of the irradiation time, and the maximum temperature reached 65°C, which shows that YB1-INPs are used for tumor treatment and supplemented with near infrared laser irradiation The temperature at the tumor site is the highest, and the treatment effect of the tumor is better. Using INPs for tumor treatment, supplemented by near infrared laser irradiation, the temperature of the tumor site is slightly increased, and the tumor treatment effect is significantly lower than YB1-INPs, while the PBS solution is not Will have a significant effect on the temperature of the tumor site.

试验例12Test Example 12

取具有相同肿瘤，且肿瘤体积相同的同种类小鼠4只，第一只小鼠注射PBS溶液，第二只小鼠注射INPs溶液，第三只小鼠注射YB1-INPs溶液，第四只小鼠注射YB1-INPs溶液，其中4只小鼠的注射量相同，且第一只小鼠、第二只小鼠和第四只小鼠在注射12h和72h，分别采用近红外激光照射5min，(808纳米，1.18W/cm ²)，四只小鼠在不同时间的代表图，如图24所示，其中，PBS(++)代表第一只小鼠，INPs(++)代表第二只小鼠，YB1-INPs代表第三只小鼠，YB1-INPs(++)代表第四只小鼠，虚线所圈出区域为肿瘤部位。 Take 4 mice of the same type with the same tumor and the same tumor volume. The first mouse was injected with PBS solution, the second mouse was injected with INPs solution, the third mouse was injected with YB1-INPs solution, and the fourth mouse Rats were injected with YB1-INPs solution, of which 4 mice had the same injection volume, and the first mouse, the second mouse and the fourth mouse were injected with near-infrared laser for 5 min at 12 h and 72 h, respectively ( 808 nm, 1.18W/cm ² ), four mice at different times, as shown in Figure 24, where PBS(++) represents the first mouse and INPs(++) represents the second In mice, YB1-INPs represent the third mouse, YB1-INPs(++) represent the fourth mouse, and the area enclosed by the dotted line is the tumor site.

从图24可以看出，第一只小鼠在14天内，肿瘤部位持续增大，且在第21天死亡，这说明PBS溶液对肿瘤没有任何治疗作用；第二只小鼠在14天内，肿瘤部位有所增大，且在第21天死亡，这说明INPs对肿瘤没有明显的治疗作用；第三只小鼠在0-7天内，肿瘤部位明显减小，但是在7-28天，肿瘤部位又出现反弹，继续增大，但是小鼠未必死亡，这说明YB1-INPs对肿瘤具有一定治疗作用，但是无法彻底根治肿瘤；第四只小鼠在第7天肿瘤部位全部消失，且到第28天并未重新出现肿瘤，这说明采用YB1-INPs并辅以近红外激光照射，能够彻底根除肿瘤，并不复发。It can be seen from Fig. 24 that the tumor area of the first mouse continued to increase within 14 days and died on the 21st day, indicating that the PBS solution had no therapeutic effect on the tumor; the second mouse within 14 days, the tumor The site has increased and died on the 21st day, indicating that INPs have no obvious therapeutic effect on the tumor; the third mouse had a significant decrease in the tumor site in 0-7 days, but in the 7-28 days, the tumor site It rebounded and continued to increase, but the mice did not necessarily die, indicating that YB1-INPs had a certain therapeutic effect on the tumor, but the tumor could not be completely cured; the fourth mouse disappeared on the 7th day and all the tumors disappeared on the 28th day. The tumor did not reappear in the sky, which means that YB1-INPs combined with near infrared laser irradiation can completely eradicate the tumor without recurrence.

试验例13Test Example 13

取具有相同肿瘤，且肿瘤体积相同的同种类小鼠20只，分成4组，每组5只，第一组小鼠注射PBS溶液，第二组小鼠注射INPs溶液，第三组小鼠注射YB1-INPs溶液，第四组小鼠注射YB1-INPs溶液，其中四组小鼠的注射量相同，且第一组小鼠、第二组小鼠和第四组小鼠在注射12h和72h，分别采用近红外激光照射5min(808纳米，1.18W/cm ²)，分别检测四组小鼠的肿瘤平均体积，并绘制四组小鼠肿瘤平均体积随时间的关系曲线，如图25所示，其中，PBS(++)代表第一组小鼠，INPs(++)代表第二组小鼠，YB1-INPs代表第三组小鼠，YB1-INPs(++)代表第四组小鼠。 Twenty mice of the same type with the same tumor and the same tumor volume were divided into 4 groups of 5 mice. The first group of mice was injected with PBS solution, the second group of mice was injected with INPs solution, and the third group of mice was injected. YB1-INPs solution, the fourth group of mice was injected with YB1-INPs solution, in which the four groups of mice were injected the same amount, and the first group of mice, the second group of mice and the fourth group of mice were injected at 12h and 72h, Using near-infrared laser irradiation for 5 min (808 nm, 1.18 W/cm ² ), the average tumor volume of the four groups of mice was detected, and the average tumor volume of the four groups of mice was plotted over time, as shown in Figure 25. Among them, PBS(++) represents the first group of mice, INPs(++) represents the second group of mice, YB1-INPs represents the third group of mice, and YB1-INPs(++) represents the fourth group of mice.

从图25可以看出，第四组小鼠的肿瘤体积从第4天即消失，这说明第四组小鼠的肿瘤全部治愈，且在28天未复发；第三组小鼠的肿瘤体积在8天时降低到最小值，然后随着时间的延长，肿瘤体积出现反弹，且持续增大；第二组小鼠的肿瘤体积随着时间的延长持续增大，在第20天时，小鼠全部死亡；第一组小鼠的肿瘤体积随着时间的延长持续增大，且增大速率高于第一组，在第18天，小鼠全部死亡。这说明PBS对肿瘤没有治疗作用；INPs辅以远红外激光照射时，能够减缓肿瘤的生长速度，但是不能抑制肿瘤的生长；而YB1-INPs能够有效***，缩小肿瘤体积，但是后期会出现肿瘤复发；而YB1-INPs辅以远红外激光照射时，能够彻底根治肿瘤，且不复发。As can be seen from Figure 25, the tumor volume of the mice in the fourth group disappeared from the fourth day, which means that the tumors of the mice in the fourth group were completely cured and did not recur at 28 days; At 8 days, it decreased to the minimum value, then with the extension of time, the tumor volume rebounded and continued to increase; the tumor volume of the second group of mice continued to increase with time, and on the 20th day, all the mice died The tumor volume of the first group of mice continued to increase with time, and the increase rate was higher than that of the first group. On the 18th day, all the mice died. This shows that PBS has no therapeutic effect on tumors; INPs supplemented with far-infrared laser irradiation can slow down the growth rate of tumors, but cannot inhibit tumor growth; and YB1-INPs can effectively treat tumors and reduce tumor volume, but tumors will appear in later stages. Relapse; while YB1-INPs supplemented with far-infrared laser irradiation can completely cure the tumor without recurrence.

试验例14Test Example 14

分别统计试验例13中不同时间时4组小鼠的生存率，并绘制小鼠生存率和时间的关系曲线，如图26所示，其中，PBS(++)代表第一组小鼠，INPs(++)代表第二组小鼠，YB1-INPs代表第三组小鼠，YB1-INPs(++)代表第四组小鼠。Count the survival rates of the four groups of mice at different times in Test Example 13, and plot the relationship between the survival rates of the mice and the time, as shown in Figure 26, where PBS(++) represents the first group of mice, INPs (++) represents the second group of mice, YB1-INPs represents the third group of mice, and YB1-INPs (++) represents the fourth group of mice.

从图26可以看出，第一组小鼠在第16天时死亡小鼠1只，死亡率为20％，在第18天时，又死亡小鼠3只，死亡率达到80％，至第24天，小鼠全部死亡，死亡率达到100％；第二组小鼠在第20天时，死亡小鼠3只，死亡率60％，至第22天时，小鼠全部死亡，死亡率达到100％；第三组小鼠在第28天时，死亡小鼠2只，死亡率为40％；第四组小鼠至28天时，无小鼠死亡，死亡率为0％。这说明PBS对肿瘤没有治疗作用，在24天小鼠全部死亡；INPs辅以远红外激光照射时，能够减缓肿瘤的生长速度，但是不能抑制肿瘤的生长，在第22天时，小鼠全部死亡；而YB1-INPs能够有效***，缩小肿瘤体积，但是后期会出现肿瘤复发，在第28天时，小鼠死亡率为40％；而YB1-INPs辅以远红外激光照射时，能够彻底根治肿瘤，在第28天时，小鼠死亡率为0％。It can be seen from Fig. 26 that the first group of mice died on day 16 with 1 mouse, with a mortality rate of 20%, and on day 18, died with 3 mice, with a mortality rate of 80%, to day 24 , All mice died, the mortality rate reached 100%; the second group of mice died on the 20th day, 3 mice, a mortality rate of 60%, by the 22nd day, all the mice died, the mortality rate reached 100%; On the 28th day, the three groups of mice had 2 dead mice with a mortality rate of 40%; the fourth group had no mice died on the 28th day and the mortality rate was 0%. This shows that PBS has no therapeutic effect on tumors, and all mice died in 24 days; when INPs were supplemented with far-infrared laser irradiation, they could slow the growth rate of tumors, but they could not inhibit the growth of tumors. On day 22, all mice died. YB1-INPs can effectively treat tumors and reduce tumor volume, but tumor recurrence will occur in the later stage. On the 28th day, the mouse mortality rate is 40%; while YB1-INPs supplemented with far-infrared laser irradiation can completely cure the tumor. At day 28, the mouse mortality rate was 0%.

试验例15Test Example 15

取具有相同肿瘤，且肿瘤体积相同的同种类小鼠2只，第一只小鼠注射PBS溶液，第二只小鼠注射YB1-INPs溶液，其中，2只小鼠注射量相同，且第二只小鼠在注射12h，采用近红外激光照射5分钟(808纳米，1.18W/cm ²)。 Two mice of the same type with the same tumor and the same tumor volume were taken. The first mouse was injected with PBS solution and the second mouse was injected with YB1-INPs solution. Among them, the two mice were injected with the same amount and the second The mice were injected with a near-infrared laser for 5 minutes (808 nm, 1.18 W/cm ² ) at 12 hours after injection.

在注射72h后，分别将第一只小鼠和第二只小鼠处死，并取出心、肝、脾、肺和肾脏组织进行染色，结果如图27所示，其中，PBS代表第一只小鼠，YB1-INPs(+)代表第二只小鼠。从图27可以看出，第一只小鼠和第二只小鼠心、肝、脾、肺和肾脏组织进行染色无明显差别，这说明实施例8提供的YB1-INPs不会对小鼠体内组织造成损伤，其在体内的安全性良好。72h after injection, the first mouse and the second mouse were sacrificed, and the heart, liver, spleen, lung and kidney tissues were removed and stained. The results are shown in Figure 27, where PBS represents the first small Mouse, YB1-INPs(+) represents the second mouse. As can be seen from Figure 27, there is no significant difference in staining the heart, liver, spleen, lung and kidney tissues of the first and second mice, which shows that the YB1-INPs provided in Example 8 will not The tissue causes damage and its safety in the body is good.

最后应说明的是：以上各实施例仅用以说明本申请的技术方案，而非对其限制；尽管参照前述各实施例对本申请进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本申请各实施例技术方案的范围。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features can be equivalently replaced; and these modifications or replacements do not deviate from the essence of the corresponding technical solutions of the technical solutions of the embodiments of the present application. range.

Claims

一种细菌-光热纳米颗粒复合物，其特征在于，包括光热纳米颗粒和细菌，所述细菌与所述光热纳米颗粒通过化学键连接；A bacteria-photothermal nanoparticle composite, characterized in that it includes photothermal nanoparticles and bacteria, and the bacteria and the photothermal nanoparticles are connected by a chemical bond;

优选地，所述细菌和所述光热纳米颗粒通过共价键连接。Preferably, the bacteria and the photothermal nanoparticles are connected by a covalent bond.
根据权利要求1所述的细菌-光热纳米颗粒复合物，其特征在于，所述细菌选自沙门氏菌、李斯特菌、大肠埃希氏菌和乳酸菌中的至少一种，优选为沙门氏菌，进一步优选为减毒沙门氏菌，更进一步优选为YB1；The bacteria-photothermal nanoparticle composite according to claim 1, characterized in that the bacteria are at least one selected from the group consisting of Salmonella, Listeria, Escherichia coli and lactic acid bacteria, preferably Salmonella, further preferably For attenuated Salmonella, YB1 is further preferred;

和/或，所述光热纳米颗粒包括光敏剂和纳米颗粒，所述光敏剂负载于所述纳米颗粒上，所述光敏剂选自ICG、PpIX和Ce6中的至少一种，优选为ICG；And/or, the photothermal nanoparticles include photosensitizers and nanoparticles, the photosensitizers are supported on the nanoparticles, and the photosensitizers are selected from at least one of ICG, PpIX and Ce6, preferably ICG;

优选地，所述纳米颗粒选自磷脂聚合物纳米颗粒、PLGA、脂质体、金纳米笼、金纳米棒和介孔硅中的至少一种，优选为PLGA和磷脂聚合物纳米颗粒；Preferably, the nanoparticles are selected from at least one of phospholipid polymer nanoparticles, PLGA, liposomes, gold nanocages, gold nanorods and mesoporous silicon, preferably PLGA and phospholipid polymer nanoparticles;

优选地，所述光热纳米颗粒的粒径为40-200nm，优选为50-100nm。Preferably, the particle diameter of the photothermal nanoparticles is 40-200nm, preferably 50-100nm.
根据权利要求1所述的细菌-光热纳米颗粒复合物，其特征在于，所述光热纳米颗粒包载有药物，所述药物包括化疗药物和生物活性分子；The bacteria-photothermal nanoparticle composite according to claim 1, wherein the photothermal nanoparticles contain a drug, and the drug includes a chemotherapy drug and a biologically active molecule;

优选地，所述化疗药物包括阿霉素类化疗药物、铂类化疗药、紫杉醇类化疗药物、IDO抑制剂、寡核苷酸DNA和脂多糖中的至少一种；Preferably, the chemotherapy drugs include at least one of adriamycin-based chemotherapy drugs, platinum-based chemotherapy drugs, paclitaxel-based chemotherapy drugs, IDO inhibitors, oligonucleotide DNA and lipopolysaccharide;

优选地，所述生物活性分子包括免疫检查点蛋白抑制剂、免疫激动剂、干扰素和白介素中的至少一种；Preferably, the biologically active molecules include at least one of immune checkpoint protein inhibitors, immune agonists, interferons and interleukins;

优选地，所述免疫检查点蛋白抑制剂包括PD-1单克隆抗体、PD-L1单克隆抗体、CTLA-4单克隆抗体、LAG-3单克隆抗体、TIM3单克隆抗体、TIGHT单克隆抗体和VISTA单克隆抗体中的至少一种；Preferably, the immune checkpoint protein inhibitors include PD-1 monoclonal antibody, PD-L1 monoclonal antibody, CTLA-4 monoclonal antibody, LAG-3 monoclonal antibody, TIM3 monoclonal antibody, TIGHT monoclonal antibody and At least one of VISTA monoclonal antibodies;

优选地，所述免疫激动剂包括4-1BB单克隆抗体和/或STING单克隆抗体。Preferably, the immune agonist comprises 4-1BB monoclonal antibody and/or STING monoclonal antibody.
根据权利要求1-3任一项所述的细菌-光热纳米颗粒复合物，其特征在于，所述复合物包括YB1和光热纳米颗粒，所述YB1和所述光热纳米颗粒通过化学键连接，所述光热纳米颗粒包括ICG、PLGA和磷脂聚合物纳米颗粒；The bacteria-photothermal nanoparticle composite according to any one of claims 1 to 3, wherein the composite comprises YB1 and photothermal nanoparticles, and the YB1 and the photothermal nanoparticles are connected by a chemical bond , The photothermal nanoparticles include ICG, PLGA and phospholipid polymer nanoparticles;

优选地，所述ICG和所述PLGA包裹于所述磷脂聚合物纳米颗粒的内部，所述磷脂聚合物纳米颗粒和所述YB1通过化学键连接；Preferably, the ICG and the PLGA are wrapped inside the phospholipid polymer nanoparticles, and the phospholipid polymer nanoparticles and the YB1 are connected by a chemical bond;

进一步优选地，所述ICG负载于所述PLGA上。Further preferably, the ICG is loaded on the PLGA.
一种细菌-光热纳米颗粒复合物的制备方法，其特征在于，包括如下步骤：将细菌和光热纳米颗粒混合，使得细菌和光热纳米颗粒通过化学键连接，即得到细菌-光热纳米颗粒复合物。A preparation method of bacteria-photothermal nanoparticle composites, characterized in that it includes the following steps: mixing bacteria and photothermal nanoparticles, so that bacteria and photothermal nanoparticles are connected by chemical bonds to obtain bacteria-photothermal nanoparticles Complex.
根据权利要求5所述的制备方法，其特征在于，包括如下步骤：The preparation method according to claim 5, comprising the following steps:

将细菌溶液、光热纳米颗粒和偶联剂混合均匀，使得细菌和光热纳米颗粒通过共价键连接，得到纳米颗粒复合物；Mix the bacterial solution, the photothermal nanoparticles and the coupling agent uniformly, so that the bacteria and the photothermal nanoparticles are connected by a covalent bond to obtain a nanoparticle composite;

优选地，所述细菌选自沙门氏菌、李斯特菌、大肠埃希氏菌和乳酸菌中的至少一种，优选为沙门氏菌，进一步优选为减毒沙门氏菌，更进一步优选为YB1；Preferably, the bacteria is selected from at least one of Salmonella, Listeria, Escherichia coli, and lactic acid bacteria, preferably Salmonella, further preferably attenuated Salmonella, even more preferably YB1;

优选地，所述光热纳米颗粒包括光敏剂和纳米颗粒，所述光敏剂负载于所述纳米颗粒上；所述光敏剂选自ICG、PpIX和Ce6中的至少一种，优选为ICG；所述纳米颗粒选自磷脂聚合物纳米颗粒、PLGA、脂质体、金纳米笼、金纳米棒和介孔硅中的至少一种，优选为磷脂聚合物纳米颗粒和PLGA；Preferably, the photothermal nanoparticles include photosensitizer and nanoparticles, and the photosensitizer is supported on the nanoparticles; the photosensitizer is selected from at least one of ICG, PpIX and Ce6, preferably ICG; The nanoparticles are selected from at least one of phospholipid polymer nanoparticles, PLGA, liposomes, gold nanocages, gold nanorods and mesoporous silicon, preferably phospholipid polymer nanoparticles and PLGA;

优选地，所述偶联剂为EDC和/或NHS。Preferably, the coupling agent is EDC and/or NHS.
根据权利要求6所述的制备方法，其特征在于，所述光热纳米颗粒和所述细菌溶液的质量比为(0.5-1.5):10，优选为1:10；The preparation method according to claim 6, wherein the mass ratio of the photothermal nanoparticles and the bacterial solution is (0.5-1.5): 10, preferably 1:10;

优选地，所述细菌溶液的菌落数为(1-2)×10 ⁸cfu/mL，优选为1×10 ⁸cfu/mL。 Preferably, the bacterial solution has a colony number of (1-2)×10 ⁸ cfu/mL, preferably 1×10 ⁸ cfu/mL.
根据权利要求5所述的制备方法，其特征在于，所述光热纳米颗粒为ICG-PLGA-磷脂聚合物纳米颗粒，所述ICG-PLGA-磷脂聚合物纳米颗粒的制备方法包括如下步骤：The preparation method according to claim 5, wherein the photothermal nanoparticles are ICG-PLGA-phospholipid polymer nanoparticles, and the preparation method of the ICG-PLGA-phospholipid polymer nanoparticles includes the following steps:

(a)将大豆卵磷脂溶液、DSPE-PEG-COOH溶液和ICG溶液混合，得到混合溶液；(a) Mix the soybean lecithin solution, DSPE-PEG-COOH solution and ICG solution to obtain a mixed solution;

(b)将PLGA溶液在超声条件下加入上述混合溶液中，得到ICG-PLGA-磷脂聚合物纳米颗粒；其中，ICG-PLGA-磷脂聚合物纳米颗粒中，ICG和PLGA包裹于磷脂聚合物纳米颗粒的内部。(b) Add the PLGA solution to the above mixed solution under ultrasonic conditions to obtain ICG-PLGA-phospholipid polymer nanoparticles; wherein, in ICG-PLGA-phospholipid polymer nanoparticles, ICG and PLGA are wrapped in phospholipid polymer nanoparticles internal.
根据权利要求8所述的制备方法，其特征在于，PLGA、大豆卵磷脂、DSPE-PEG-COOH和ICG的质量比为(30-35):(2-4):(1-5):(10-17)，优选为33:3:2:15。The preparation method according to claim 8, wherein the mass ratio of PLGA, soybean lecithin, DSPE-PEG-COOH and ICG is (30-35):(2-4):(1-5):( 10-17), preferably 33:3:2:15.
根据权利要求1-4任一项所述的细菌-光热纳米颗粒复合物或根据权利要求5-9任一项所述的制备方法得到的细菌-光热纳米颗粒复合物在制备肿瘤治疗药物中的应用。The bacterial-photothermal nanoparticle complex according to any one of claims 1 to 4 or the bacterial-photothermal nanoparticle complex obtained according to the preparation method according to any one of claims 5 to 9 in the preparation of a tumor therapeutic drug Application.