技术领域technical field
本发明涉及一种多级靶向递送、分步药物控释的抗肿瘤纳米气体药物，其制备方法及其用途，属于纳米材料技术领域和生物医学领域。The invention relates to an anti-tumor nano-gas drug with multi-level targeted delivery and step-by-step controlled release of drugs. The preparation method and application thereof belong to the technical field of nanomaterials and the field of biomedicine.
背景技术Background technique
众所周知，吸入过量的一氧化碳(CO)会引起血红蛋白的携氧能力下降，从而引起中毒。但是，低浓度的CO却可以在与多价过渡金属特异性结合的过程中扮演信使的角色，从而调节神经系统、心血管系统和免疫系统等大量系统的多种生理功能。CO治疗主要具有四种作用机制：免疫系统调节；细胞保护；血管舒张活性响应；氧化还原调控。因而，CO在抗癌、防止移植器官的缺血性坏死和血液再灌注损伤、抗心血管病等方面具有非常显著的治疗活性。It is well known that inhalation of excessive carbon monoxide (CO) can cause the oxygen-carrying capacity of hemoglobin to decrease, thereby causing poisoning. However, low concentrations of CO can play the role of a messenger in the process of specifically binding to multivalent transition metals, thereby regulating a variety of physiological functions in a large number of systems such as the nervous system, cardiovascular system, and immune system. CO therapy has four main mechanisms of action: modulation of the immune system; cytoprotection; response to vasodilation activity; and redox regulation. Therefore, CO has very significant therapeutic activity in anti-cancer, prevention of ischemic necrosis and blood reperfusion injury of transplanted organs, and anti-cardiovascular disease.
特别是在抗癌方面，最近的研究结果表明：CO的主要作用靶点在细胞线粒体，CO可以通过快速耗尽癌细胞的生物能，加速癌细胞的凋亡并阻止癌细胞的繁殖，即抗沃伯格效应；同时，CO还会抑制正常细胞的能量代谢，从而选择性地保护正常细胞的活性和生理功能。然而，临床上气体治疗的两种给药方式——直接吸入治疗性气体和注射能够释放气体的分子化合物(气体前药)，均难以控制气体的血药浓度和病灶区的有效浓度。这主要归因于这些给药方式都无法实现靶向性CO输送和CO的可控释放。过高的CO血药浓度将引起中毒(>20％CO-Hb)，但过低的CO血药浓度无法满足病灶部位的局部CO需求量。理想的安全且有效的气体治疗策略是：既保证治疗性气体特异性高浓度蓄积于病灶部位、又保证治疗性气体在血液中保持较低的浓度。因此，实现CO气体的靶向传输和可控释放对于提高CO药效、规避CO中毒风险至关重要，对于发展气体治疗用于抗肿瘤研究具有重要的应用价值。Especially in terms of anti-cancer, recent research results show that: the main target of CO is in the mitochondria of cells. CO can accelerate the apoptosis of cancer cells and prevent the proliferation of cancer cells by rapidly depleting the bioenergy of cancer cells, that is, anti-cancer cells. Warburg effect; at the same time, CO can also inhibit the energy metabolism of normal cells, thereby selectively protecting the activity and physiological functions of normal cells. However, it is difficult to control the blood drug concentration of the gas and the effective concentration of the lesion in the two clinical administration methods of gas therapy—direct inhalation of therapeutic gas and injection of molecular compounds (gas prodrugs) that can release gas. This is mainly attributed to the inability of these delivery methods to achieve targeted CO delivery and controlled release of CO. Too high plasma concentration of CO will cause poisoning (>20% CO-Hb), but too low plasma concentration of CO cannot meet the local CO demand of the lesion. The ideal safe and effective gas therapy strategy is to ensure that the specific high concentration of therapeutic gas is accumulated in the lesion, and to ensure that the concentration of therapeutic gas in the blood is kept low. Therefore, realizing the targeted delivery and controllable release of CO gas is crucial to improving the efficacy of CO and avoiding the risk of CO poisoning, and has important application value for the development of gas therapy for anti-tumor research.
发明内容Contents of the invention
鉴于以上内容，有必要提供一种连续靶向肿瘤组织─肿瘤细胞─细胞内线粒体的纳米药物，用于CO气体的靶向传输和高效肿瘤治疗。我们设计合成了一种多级靶向和多步响应性释放CO的正电性新型CORMs前药(简称MeCO-Mito)，利用负电性介孔二氧化硅纳米颗粒作为药物载体(简称MSN)通过静电吸附和毛细作用吸附CO前药(MeCO-Mito@MSN)，进一步包裹肿瘤细胞靶向分子透明质酸(简称HA)，构建一种具有靶向肿瘤细胞线粒体并原位控释CO功能的新型智能纳米药物(MeCO-Mito@MSN@HA)。结合靶向输送技术和纳米药物可控释放技术，最终达到CO在肿瘤细胞线粒体部位靶向传输和可控释放的目的，实现安全、高效、可控的CO治疗。本发明还进一步提供一种上述纳米药物的制备方法及应用。In view of the above, it is necessary to provide a nano drug that continuously targets tumor tissue-tumor cells-intracellular mitochondria for targeted delivery of CO gas and efficient tumor treatment. We designed and synthesized a new type of positively charged CORMs prodrug (MeCO-Mito) with multi-stage targeting and multi-step responsive release of CO, using negatively charged mesoporous silica nanoparticles as drug carriers (MSN for short) through Electrostatic adsorption and capillary action adsorbed CO prodrug (MeCO-Mito@MSN), and further encapsulated the tumor cell targeting molecule hyaluronic acid (HA for short), constructing a new type of drug with the function of targeting tumor cell mitochondria and in situ controlled release of CO Smart nanomedicine (MeCO-Mito@MSN@HA). Combining the targeted delivery technology and the controlled release technology of nano-drugs, the goal of targeted delivery and controlled release of CO in the mitochondria of tumor cells can be finally achieved, and a safe, efficient and controllable CO treatment can be realized. The present invention further provides a preparation method and application of the above-mentioned nanomedicine.
一种肿瘤靶向纳米药物，所述的肿瘤靶向纳米药物通过三级靶向给药和二级CO控释实现对肿瘤进行针对性的治疗；A tumor-targeting nano-medicine, the tumor-targeting nano-medicine achieves targeted treatment of tumors through tertiary targeted drug delivery and secondary CO controlled release;
所述的三级靶向包括有：所述的纳米药物通过EPR效应在肿瘤组织内蓄积的第一级靶向、通过对肿瘤细胞内的蛋白靶向识别进行特异性结合实现药物进一步传输的第二级靶向和纳米药物释放的CO前药进入细胞线粒体内并在线粒体内蓄积的第三级靶向；The third-level targeting includes: the first-level targeting of the nano-medicine accumulated in the tumor tissue through the EPR effect, and the second-level targeting of the further delivery of the drug through specific binding to the protein target recognition in the tumor cells. Secondary targeting and tertiary targeting in which the CO prodrug released by the nanomedicine enters the mitochondria of the cell and accumulates in the mitochondria;
所述的二级CO控释包括有：通过正电性CO前药与酸性环境的质子交换来实现酸响应性CO前药控释的第一级控释和CO前药在线粒体内ROS的作用下响应性控释CO的第二级控释。The two-stage CO controlled release includes: the first-stage controlled release of the acid-responsive CO prodrug controlled release through the proton exchange between the positively charged CO prodrug and the acidic environment, and the role of the CO prodrug in the mitochondria of ROS Second-level controlled release of lower responsive controlled release of CO.
优选的，所述的CO前药由CO施体和线粒体靶向分子键合而成。所述CO施体包括但不仅限于金属羰基化合物及其衍生物，如羰基铁、羰基锰和羰基钌；所述线粒体靶向分子包括但不仅限于三苯基膦、罗丹明、4-[(E)-2-(1H-吲哚-3-基)乙烯基]-1-甲基吡啶鎓碘化物、胍盐、地喹氯铵及其衍生物；所述的CO前药与线粒体膜有高度亲和性，具备线粒体靶向功能。Preferably, the CO prodrug is formed by bonding a CO donor and a mitochondrial targeting molecule. The CO donor includes but not limited to metal carbonyl compounds and their derivatives, such as carbonyl iron, carbonyl manganese and carbonyl ruthenium; the mitochondrial targeting molecules include but not limited to triphenylphosphine, rhodamine, 4-[(E )-2-(1H-indol-3-yl)vinyl]-1-picoline iodide, guanidinium salt, dequalinium chloride and derivatives thereof; the CO prodrug has a high degree of Affinity, with mitochondrial targeting function.
一种肿瘤靶向纳米药物，所述的纳米药物由介孔二氧化硅纳米颗粒装载所述的CO前药并在颗粒外部包裹透明质酸靶向分子，具有肿瘤组织-肿瘤细胞-线粒体多级靶向层层推进的功能和在线粒体内CO控释功能。一种肿瘤组织-肿瘤细胞-线粒体多级靶向功能和线粒体内CO控释功能的抗肿瘤纳米气体药物作为制备抗肿瘤药物的应用。A tumor-targeted nanomedicine, the nanomedicine is loaded with the CO prodrug by mesoporous silica nanoparticles and coated with hyaluronic acid targeting molecules on the outside of the particles, and has a tumor tissue-tumor cell-mitochondrion multilevel target The function of advancing layer by layer and the function of controlled release of CO in mitochondria. An anti-tumor nanometer gas drug with a tumor tissue-tumor cell-mitochondrion multilevel targeting function and a controlled release function of CO in the mitochondria is used as an anti-tumor drug.
所述的纳米药物通过静电吸附和毛细吸附，CO前药高效担载在介孔二氧化硅纳米颗粒中；通过质子交换作用实现酸控药物释放；释放的CO前药在线粒体部位响应性释放CO。The nano-drug is efficiently loaded in the mesoporous silica nanoparticles through electrostatic adsorption and capillary adsorption; the acid-controlled drug release is realized through proton exchange; the released CO prodrug responds to the mitochondrial site to release CO .
所述的纳米药物通过静电吸附和氢键作用，透明质酸靶向分子(HA)很容易包覆在纳米颗粒表面，具备肿瘤细胞靶向功能。The nano-medicine is easily coated on the surface of the nano-particle with the hyaluronic acid targeting molecule (HA) through electrostatic adsorption and hydrogen bonding, and has the function of targeting tumor cells.
本发明还提供一种肿瘤靶向的纳米药物的制备方法，其包括如下步骤：The present invention also provides a method for preparing a tumor-targeted nanomedicine, which includes the following steps:
制备所述的CO前药；preparing the CO prodrug;
制备介孔二氧化硅纳米颗粒；Preparation of mesoporous silica nanoparticles;
将CO前药装载于所述介孔二氧化硅纳米颗粒中；loading CO prodrugs in the mesoporous silica nanoparticles;
在纳米颗粒表面包裹透明质酸靶向分子，以得到所述纳米药物。Wrapping hyaluronic acid targeting molecules on the surface of nanoparticles to obtain the nanomedicine.
CO前药装载可以采用真空纳米浇铸法，所述真空纳米浇铸法的具体步骤为：将所述二氧化硅纳米颗粒分散于有机溶剂，以得到二氧化硅纳米颗粒分散液；向所述二氧化硅纳米颗粒分散液中加入CO前药，通过浸渍、离心、清洗，以得到。The CO prodrug loading can adopt the vacuum nano-casting method, and the specific steps of the vacuum nano-casting method are: disperse the silica nanoparticles in an organic solvent to obtain a silica nanoparticle dispersion; The CO prodrug is added to the silicon nanoparticle dispersion liquid, and the method is obtained by impregnating, centrifuging and washing.
本发明的有益效果在于，相较于现有技术，本发明的多级靶向递送，分步释放的抗肿瘤纳米气体药物，通过采用生物相容性好且可降解的介孔二氧化硅纳米颗粒为载体，并高效担载CO前药，以构建一种响应性靶向线粒体的CO气体治疗纳米药物。本发明的MeCO-Mito@MSN@HA纳米药物具有肿瘤部位原位响应性的释放CO前药和线粒体部位响应性释放CO气体的特性，无需外界刺激源的介导，因此，采用上述纳米药物治疗肿瘤是一种无创、绿色的治疗方式。此外，本发明的纳米药物合成工艺简单，且合成效率高。进一步地，由于药物合成的原料低廉，从而降低生产成本，因而适合大规模生产。The beneficial effect of the present invention lies in that, compared with the prior art, the anti-tumor nano-gas drug for multi-level targeted delivery and step-by-step release of the present invention is achieved through the use of biocompatible and degradable mesoporous silica nano The particles are used as carriers and efficiently loaded CO prodrugs to construct a CO gas therapeutic nanomedicine responsively targeting mitochondria. The MeCO-Mito@MSN@HA nanomedicine of the present invention has the characteristics of in situ responsive release of CO prodrug at the tumor site and responsive release of CO gas at the mitochondrial site, without the mediation of external stimuli. Therefore, the above nanomedicine treatment Tumor is a non-invasive, green treatment. In addition, the nano drug synthesis process of the present invention is simple, and the synthesis efficiency is high. Furthermore, because the raw materials for drug synthesis are cheap, the production cost is reduced, and thus it is suitable for large-scale production.
附图说明Description of drawings
图1是本发明CO前药的一较佳实施方式的合成示意图。Figure 1 is a schematic diagram of the synthesis of a preferred embodiment of the CO prodrug of the present invention.
图2-3是本发明CO前药，中间产物和原料的核磁，质谱和红外的表征图。Figures 2-3 are NMR, mass spectrometry and infrared characterization diagrams of CO prodrugs, intermediates and raw materials of the present invention.
图4是为制备FeCO-TPP@MSN@HA纳米药物的合成流程图。Figure 4 is a synthesis flow chart for the preparation of FeCO-TPP@MSN@HA nanomedicine.
图5是本发明FeCO-TPP@MSN@HA纳米药物的TEM照片及对应的元素分布图。Fig. 5 is a TEM photo of the FeCO-TPP@MSN@HA nanomedicine of the present invention and the corresponding element distribution diagram.
图6是本发明的MeCO-Mito@MSN@HA纳米药物的Zeta电位,DLS粒度分析和红外光谱的表征图。Fig. 6 is a characterization diagram of the Zeta potential, DLS particle size analysis and infrared spectrum of the MeCO-Mito@MSN@HA nanomedicine of the present invention.
图7是本发明的MeCO-Mito@MSN@HA纳米药物的BET结果图。Fig. 7 is a BET result diagram of the MeCO-Mito@MSN@HA nanomedicine of the present invention.
图8是通过MeCO-Mito@MSN作用不同PH后的响应性释放COP前药以及CO前药通过不同浓度羟基自由基响应性释放CO气体的结果图。Fig. 8 is a diagram showing the responsive release of COP prodrug and CO prodrug through different concentrations of hydroxyl radicals to release CO gas after MeCO-Mito@MSN acts on different pH.
图9是通过作用正常细胞和肿瘤细胞后的响应性释放MeCO-Mito@MSN@HA纳米药物的示踪图。Figure 9 is a trace diagram of the responsive release of MeCO-Mito@MSN@HA nanomedicine after acting on normal cells and tumor cells.
图10是本发明的MeCO-Mito@MSN@HA纳米药物通过作用不同细胞的毒性测试结果图。Fig. 10 is a graph showing the toxicity test results of the MeCO-Mito@MSN@HA nanomedicine of the present invention acting on different cells.
图11是本发明的MeCO-Mito@MSN@HA纳米药物在不同细胞代谢水平上的毒性效果的评价图。Fig. 11 is an evaluation diagram of the toxicity effect of the MeCO-Mito@MSN@HA nanomedicine of the present invention at different cell metabolism levels.
图12是本发明的MeCO-Mito@MSN@HA纳米药物对不同细胞线粒体膜的影响效果的示踪图。Fig. 12 is a trace diagram of the effect of the MeCO-Mito@MSN@HA nanomedicine of the present invention on the mitochondrial membrane of different cells.
图13是本发明的MeCO-Mito@MSN@HA纳米药物进入小鼠血液，在小鼠肿瘤部位蓄积的示踪图。Fig. 13 is a trace diagram of the MeCO-Mito@MSN@HA nanomedicine of the present invention entering the mouse blood and accumulating in the tumor site of the mouse.
图14是本发明的MeCO-Mito@MSN@HA纳米药物对在动物水平上的治疗效果的评价对比图。Figure 14 is a comparison chart of the evaluation of the therapeutic effect of the MeCO-Mito@MSN@HA nanomedicine of the present invention on the animal level.
图15是小鼠经过不同模式治疗20天后主要组织器官和肿瘤的生理组织切片图。Figure 15 is a diagram of the physiological tissue sections of the main tissues, organs and tumors of the mice treated with different modes for 20 days.
具体实施方式Detailed ways
下面通过具体实施例对本发明做进一步的说明。The present invention will be further described below through specific examples.
(1)制备CO前药(1) Preparation of CO prodrug
使用Mito-COOH(带羧基的线粒体靶向基团)和2-氨基乙硫醇(简称NH2EtSH)作为反应原料，通过酯化反应，将巯基连接在具有线粒体靶向性能的Mito分子的端链上(Mito-SH)。通过巯基和合金(简称MeCO)之间的配位替换反应，将Mito-SH与MeCO中的核进行配位，合成CO前药分子。Using Mito-COOH (mitochondrial targeting group with carboxyl group) and 2-aminoethanethiol (abbreviated as NH2 EtSH) as the reaction raw materials, the sulfhydryl group is connected to the end of the Mito molecule with mitochondrial targeting properties through esterification reaction On-chain (Mito-SH). The CO prodrug molecule was synthesized by coordinating Mito-SH with the nucleus in MeCO through a coordination substitution reaction between thiol and alloy (MeCO for short).
将Mito-COOH(1mmol)溶解于2mL二甲基亚砜(DMSO)和2mL甲醇中，随后加入EDC(1.5mmol)和NHS(1.5mmol)。搅拌3h后，加入半胱胺(1.1mol)和三乙醇(80μL)，反应体系在RT下搅拌12h，用DMSO(40mL)与去离子水(10mL)的混合溶液进行萃取，用无水硫酸钠干燥，将有机相旋干即得到Mito-SH。然后，将Me(77.23mg，0.153mmol)和Mito-SH(378mg，0.926mmol)溶解于DMSO(4mL)中，然后在70℃下在氮气气氛下加热2h。加热过程中，绿色溶液逐渐变成深红色，反映出配位反应的发生，最后，用一定量的去离子水纯化红色产物，然后在冷冻下干燥。整个反应和保存都需要避光。Mito-COOH (1 mmol) was dissolved in 2 mL dimethylsulfoxide (DMSO) and 2 mL methanol, followed by the addition of EDC (1.5 mmol) and NHS (1.5 mmol). After stirring for 3 h, cysteamine (1.1 mol) and triethanol (80 μL) were added, the reaction system was stirred at RT for 12 h, extracted with a mixed solution of DMSO (40 mL) and deionized water (10 mL), and then extracted with anhydrous sodium sulfate After drying, the organic phase was spin-dried to obtain Mito-SH. Then, Me (77.23 mg, 0.153 mmol) and Mito-SH (378 mg, 0.926 mmol) were dissolved in DMSO (4 mL), then heated at 70 °C for 2 h under nitrogen atmosphere. During the heating process, the green solution gradually turned dark red, reflecting the occurrence of the coordination reaction. Finally, the red product was purified with a certain amount of deionized water, and then dried under freezer. The entire reaction and storage should be protected from light.
(2)制备介孔二氧化硅纳米颗粒(2) Preparation of mesoporous silica nanoparticles
取2g的CTAC(25wt.％)(十六烷基三甲基氯化铵)和0.02g TEAH(氢氧化四乙基铵)加入到20mL去离子水中，搅拌均匀后,升温到80℃，以一定速率加入TEOS(1.5mL)(正硅酸乙酯),滴加速率控制在0.003mL/min，继续反应1h。趁热离心后，分别水洗和醇洗1次后，加入无水甲醇的NaCl溶液萃取CTAC。最终离心水洗和醇溶液清洗，以得到介孔MSN载体。Take 2g of CTAC (25wt.%) (cetyltrimethylammonium chloride) and 0.02g TEAH (tetraethylammonium hydroxide) into 20mL of deionized water, after stirring evenly, heat up to 80 ° C to Add TEOS (1.5mL) (tetraethyl orthosilicate) at a certain rate, control the dropping rate at 0.003mL/min, and continue the reaction for 1h. After centrifugation while hot, wash with water and alcohol once respectively, add anhydrous methanol NaCl solution to extract CTAC. Finally, wash with water and alcohol solution by centrifugation to obtain the mesoporous MSN carrier.
(3)制备MeCO-Mito@MSN(3) Preparation of MeCO-Mito@MSN
首先取2mL MSN的无水甲醇溶液(2mg/mL)，然后加入6mg的CO前药，充分溶解后，置于真空干燥箱中，待溶液挥发80％左右后，离心水洗后，以制得肿瘤靶向的MeCO-Mito@MSN。在本实施例中，所述MeCO-Mito@MSN的负载量约为32.9％。First take 2mL of MSN anhydrous methanol solution (2mg/mL), then add 6mg of CO prodrug, after fully dissolving, put it in a vacuum drying oven, wait for about 80% of the solution to volatilize, centrifuge and wash with water to obtain the tumor Targeted MeCO-Mito@MSN. In this embodiment, the loading of MeCO-Mito@MSN is about 32.9%.
(4)制备MeCO-Mito@MSN@HA。(4) Preparation of MeCO-Mito@MSN@HA.
收集的MeCO-Mito@MSN在振荡下完全再分散到水中，并逐渐加入到HA的PBS溶液中，在低温下，将混合物稳定2h，然后离心收集MeCO-Mito@MSN@HA纳米颗粒，用PBS洗涤两次。The collected MeCO-Mito@MSN was completely redispersed into water under shaking, and gradually added to the PBS solution of HA. At low temperature, the mixture was stabilized for 2 h, and then the MeCO-Mito@MSN@HA nanoparticles were collected by centrifugation, and washed with PBS. Wash twice.
图1-3展示了CO前药的合成流程图以及表征图，请参照图1，图示所展示的原料Mito-COOH为TPP-COOH,中间产物为TPP-SH以及最终产物为FeCO-TPP，其结构通过1HNMR和MS光谱表征得到确认,图2和图3为中间产物和原料的核磁，质谱和红外的表征图，从红外光谱(图2C)可以看出，TPP-CO继承了TPP和羰基的特征带，分别由蓝区和黄区表示，进一步验证了FeCO-TPP的结构。Figure 1-3 shows the synthesis flow chart and characterization diagram of CO prodrug, please refer to Figure 1, the raw material Mito-COOH shown in the diagram is TPP-COOH, the intermediate product is TPP-SH and the final product is FeCO-TPP, Its structure was confirmed by 1HNMR and MS spectrum characterization. Figure 2 and Figure 3 are the NMR, mass spectrum and infrared characterization diagrams of intermediate products and raw materials. It can be seen from the infrared spectrum (Figure 2C) that TPP-CO inherits TPP and carbonyl The characteristic bands of , represented by the blue and yellow regions, respectively, further validate the structure of FeCO-TPP.
图4是为制备FeCO-TPP@MSN@HA纳米药物的合成流程图，先是通过静电吸附和毛细作用，将FeCO-TPP和MSN结合，将FeCO-TPP装载到MSN内，然后再在MSN外侧包括HA。Figure 4 is the synthesis flow chart for the preparation of FeCO-TPP@MSN@HA nanomedicine. Firstly, FeCO-TPP is combined with MSN through electrostatic adsorption and capillary action, and FeCO-TPP is loaded into MSN, and then included on the outside of MSN. ha.
图5是本发明FeCO-TPP@MSN@HA纳米药物的TEM照片及对应的元素分布图。Fig. 5 is a TEM photo of the FeCO-TPP@MSN@HA nanomedicine of the present invention and the corresponding element distribution diagram.
其中，图示(5D)表示制备的MSN的SEM照片，展示了MSN良好的分散性和均一的颗粒尺寸。图示(5A)表示为制备MSN的TEM照片，结果表明，图示(5B,5C)中从颗粒的边缘能够观察到HA已经包裹MeCO-Mito@MSN。从图5E中元素分布图可以看出，所述纳米药物中含有合金(Me)、硅(Si)、碳(C)及氧(O)元素，表明了MeCO-Mito已经包裹在MSN。Among them, the diagram (5D) represents the SEM photo of the prepared MSN, which shows the good dispersion and uniform particle size of MSN. The illustration (5A) represents the TEM photograph of the prepared MSN, and the results show that in the illustration (5B, 5C), it can be observed that HA has wrapped MeCO-Mito@MSN from the edge of the particle. It can be seen from the element distribution diagram in Figure 5E that the nanomedicine contains alloy (Me), silicon (Si), carbon (C) and oxygen (O) elements, indicating that MeCO-Mito has been wrapped in MSN.
图6展示实施例1制得的肿瘤靶向的纳米药物的Zeta，DLS和红外图。图6a展示了CO前药装载后的表面电位逆转，这是由于FeCO-TPP前药被很好地封装到MSN中，每克MSN的装载量为353.9mg FeCO-TPP(图6d,6e)。FeCO-TPP@MSN的高正电位很容易通过静电组装法使得透明质酸包裹，从而使合成的FeCO-TPP@MSN@HA纳米药物的表面电位近似为中性。图6b展示了包涂透明质酸后的FeCO-TPP@MSN@HA纳米药物尺寸和分散性的得到明显改善。此外，图6c的FTIR以及图7展示的BET图结果进一步表明，成功地将FeCO-TPP和HA涂层修饰到MSN上。Figure 6 shows the Zeta, DLS and infrared images of the tumor-targeted nanomedicine prepared in Example 1. Figure 6a demonstrates the surface potential reversal after CO prodrug loading, which is due to the well-encapsulated FeCO-TPP prodrug into MSNs with a loading of 353.9 mg FeCO-TPP per gram of MSN (Figure 6d, 6e). The high positive potential of FeCO-TPP@MSN can easily wrap hyaluronic acid through the electrostatic assembly method, so that the surface potential of the synthesized FeCO-TPP@MSN@HA nanomedicine is approximately neutral. Figure 6b shows that the size and dispersion of FeCO-TPP@MSN@HA nanomedicines coated with hyaluronic acid are significantly improved. In addition, the FTIR of Figure 6c and the BET map results presented in Figure 7 further demonstrate that FeCO-TPP and HA coatings were successfully modified onto MSNs.
从图8可以看出，本发明的实施例1制得的肿瘤靶向的纳米药物在不同的pH下响应性释放CO前药的结果图。随着pH酸性的增强，其释放CO前药的速率也加快。此外，本发明中FeCO-TPP@MSN纳米药物的在磷酸盐缓冲溶液中能够稳定存在，而在微酸的磷酸盐缓冲溶液中则能够响应性缓慢释放CO前药。因此，这种酸响应性释放CO前药能够将肿瘤区域的微酸环境作为本发明纳米药物的触发源，从而实现肿瘤区域微环境的定点响应性CO前药释放，进而有效地规避CO气体中毒的风险、并提高CO气体治疗的效率。It can be seen from FIG. 8 that the tumor-targeted nanomedicine prepared in Example 1 of the present invention releases the CO prodrug responsively at different pHs. As the acidic pH increases, the release rate of the CO prodrug also increases. In addition, the FeCO-TPP@MSN nanomedicine in the present invention can exist stably in the phosphate buffer solution, while it can slowly release the CO prodrug in the slightly acidic phosphate buffer solution. Therefore, this acid-responsive release of CO prodrug can use the slightly acidic environment of the tumor area as the trigger source of the nanomedicine of the present invention, thereby realizing the targeted release of CO prodrug in the microenvironment of the tumor area, thereby effectively avoiding CO gas poisoning risk and improve the efficiency of CO gas therapy.
图8中得到制得的肿瘤靶向的纳米药物通过作用不同·OH度后的响应性释放CO的结果图。将FeCO-TPP@MSN纳米药物分别分散于浓度为5uM(微摩尔/升)、10uM、20uM的·OH的磷酸盐缓冲溶液(PBS，pH＝7.4)中，并采用脱氧血红蛋白作为CO分子探针，实时采集420nm和520nm处的紫外吸光度值。本发明通过Lambert-Beer定律定量CO浓度，从而检测CO的释放量。本发明中CO前药随着·OH浓度的增加，CO的释放速率也越来越强，这意味着对更恶性肿瘤细胞有更有效的治疗效果，因为它们具有更高的能量水平。Figure 8 shows the results of the prepared tumor-targeted nano-drugs releasing CO in response to different degrees of OH. Disperse FeCO-TPP@MSN nanomedicine in 5uM (micromole/liter), 10uM, 20uM OH phosphate buffer solution (PBS, pH=7.4), and use deoxygenated hemoglobin as CO molecular probe , real-time collection of UV absorbance values at 420nm and 520nm. The present invention quantifies the CO concentration through the Lambert-Beer law, thereby detecting the release amount of CO. The CO prodrug in the present invention has an increasingly stronger release rate of CO as the concentration of OH increases, which means a more effective therapeutic effect on more malignant tumor cells because of their higher energy level.
图9展示了通过共聚焦激光扫描显微镜(CLSM)成像，进一步评价了体外多级靶向给药和细胞内控释过程。与荧光标记的FeCO-TPP@MSN@HA灭菌处理后，再将浓度为100ug/mL的FeCO-TPP@MSN@HA分别与正常细胞和肿瘤细胞共培养，培养1，2，3，4h后，洗去培养液中的纳米药物。然后，在细胞培养基中加入细胞核探针(DAPI),分组加入溶酶体探针，线粒体探针，CO荧光探针COP-1，培养不同时间后，利用共聚焦显微镜在明视野和暗视野(荧光成像区)定性检测细胞多级靶向给药和细胞内控释过程。所述正常细胞采用人体肾脏细胞(HEK-293T细胞)和人乳腺细胞(MCF-10A细胞),所述肿瘤细胞采用小鼠黑色素瘤细胞(B16细胞),小鼠乳腺癌细胞(4T1细胞)，人宫颈癌细胞(HeLa细胞)。Figure 9 shows the imaging by confocal laser scanning microscope (CLSM) to further evaluate the in vitro multistage targeted drug delivery and intracellular controlled release process. After being sterilized with fluorescently labeled FeCO-TPP@MSN@HA, FeCO-TPP@MSN@HA at a concentration of 100ug/mL was co-cultured with normal cells and tumor cells respectively, and after 1, 2, 3, 4 hours of culture , to wash away the nanomedicine in the culture medium. Then, the cell nucleus probe (DAPI) was added to the cell culture medium, and the lysosome probe, mitochondrial probe, and CO fluorescent probe COP-1 were added in groups. (Fluorescence imaging area) Qualitative detection of multi-stage targeted drug delivery and intracellular controlled release process of cells. The normal cells are human kidney cells (HEK-293T cells) and human breast cells (MCF-10A cells), the tumor cells are mouse melanoma cells (B16 cells), mouse breast cancer cells (4T1 cells), Human cervical cancer cells (HeLa cells).
从图9中可以看出实施例1制得的肿瘤靶向的纳米药物与正常细胞和肿瘤细胞共培养一定的时间后，FeCO-TPP@MSN@HA纳米药物大量被肿瘤细胞吞噬。然而，在正常细胞的示踪图中，基本观测不到红光荧光，这表明所述纳米药物在正常细胞环境中没有可识别HA的CD44蛋白。因此，本发明的纳米药物具有肿瘤细胞的靶向性。It can be seen from Figure 9 that after the tumor-targeted nanomedicine prepared in Example 1 was co-cultured with normal cells and tumor cells for a certain period of time, a large amount of FeCO-TPP@MSN@HA nanomedicine was phagocytized by tumor cells. However, in the tracer diagram of normal cells, red light fluorescence is basically not observed, which indicates that the nanomedicine has no CD44 protein that can recognize HA in the normal cell environment. Therefore, the nanomedicine of the present invention has tumor cell targeting.
图9展示了在HeLa细胞对FeCO-TPP@MSN@HA纳米医学的细胞内吞过程,从图中发现,绿色和红色合并为黄色，纳米药物内吞进入细胞溶酶体，这由纳米颗粒的性质。此外,蓝色逐渐出现,表明CO前药从纳米颗粒中持续释放。溶酶体中的前药释放由于上述的酸响应性的释放能力。Figure 9 shows the endocytosis process of FeCO-TPP@MSN@HA nanomedicine in HeLa cells. From the figure, it is found that the green and red merge into yellow, and the nanomedicine is endocytosed into the cell lysosome, which is caused by the nanoparticle nature. In addition, blue color gradually appeared, indicating the sustained release of CO prodrug from nanoparticles. Prodrug release in lysosomes is due to the acid-responsive release capability described above.
图9中为了跟踪前药的线粒体内靶向,细胞与纳米药物共培育后,用绿色线粒体染料定位细胞线粒体。MSN载体和CO药物分别嫁接红色RITC和蓝色QL(8-氨基喹啉)的荧光,用于构建FeCO-TPP@MSN@HA纳米药物进行荧光追踪。为了更易区分药物释放的情况,共聚焦图像中的线粒体和MSN通道分别设置为红色和绿色。红色和蓝色重叠后产生了紫色,代表了前药在线粒体内的积累。紫色的增加意味着释放的CO前药在线粒体中的吸收增加。随着溶酶体纳米药物中的CO前药的释放,通过Mito基团识别,前药逐渐积累到线粒体中,显示出线粒体靶向的前药传递。In Figure 9, in order to track the intramitochondrial targeting of the prodrug, after the cells were co-cultivated with the nanomedicine, the mitochondria of the cells were localized with a green mitochondrial dye. MSN carrier and CO drug were grafted with the fluorescence of red RITC and blue QL (8-aminoquinoline), respectively, for the construction of FeCO-TPP@MSN@HA nanomedicine for fluorescence tracking. To make it easier to distinguish the drug release, the mitochondrial and MSN channels in the confocal images were set to red and green, respectively. Red and blue overlap to produce purple, representing the accumulation of the prodrug in the mitochondria. An increase in purple color means increased uptake of the released CO prodrug in the mitochondria. With the release of the CO prodrug in the lysosomal nanomedicine, the prodrug was gradually accumulated into the mitochondria through Mito group recognition, showing mitochondria-targeted prodrug delivery.
图9中荧光COP-1探针用于检测细胞内CO的释放。从图中看,随着孵育时间的增加,绿色荧光强度的增强反映了CO前药在细胞中CO的释放增加。可以发现,一旦CO前药到达HeLa细胞线粒体，线粒体内部的ROS行为引发CO的释放。上述的四个方面结果也一致证实了实例1所示的多级靶向递送，分步释放纳米药物的假设。The fluorescent COP-1 probe in Figure 9 was used to detect intracellular CO release. It can be seen from the figure that with the increase of incubation time, the enhancement of green fluorescence intensity reflects the increase of CO release from CO prodrug in cells. It can be found that once the CO prodrug reaches the mitochondria of HeLa cells, the ROS behavior inside the mitochondria triggers the release of CO. The results of the above four aspects also consistently confirmed the hypothesis of multi-stage targeted delivery and step-by-step release of nanomedicine shown in Example 1.
图10展示了实施例1制得的肿瘤靶向的纳米药物通过作用不同细胞的毒性测试结果图。将实施例1制备的FeCO-TPP@MSN@HA调成不同浓度(12.5ug/mL、25ug/mL、50ug/mL、100ug/mL)，并灭菌处理，将灭菌后的不同浓度的FeCO-TPP@MSN@HA分别与多种细胞共培养24h,36h,72h，再评价不同浓度材料对细胞的毒性实验。所述的正常细胞采用人体肾脏细胞(HEK-293T细胞)和人乳腺细胞(MCF-10A细胞),所述肿瘤细胞采用小鼠乳腺癌细胞(4T1细胞)，人宫颈癌细胞(HeLa细胞)。FIG. 10 shows the results of the toxicity test of the tumor-targeted nanomedicine prepared in Example 1 by acting on different cells. The FeCO-TPP@MSN@HA prepared in Example 1 was adjusted to different concentrations (12.5ug/mL, 25ug/mL, 50ug/mL, 100ug/mL), and sterilized, and the sterilized different concentrations of FeCO -TPP@MSN@HA was co-cultured with various cells for 24h, 36h, and 72h, and then evaluated the toxicity of different concentrations of materials to the cells. The normal cells are human kidney cells (HEK-293T cells) and human breast cells (MCF-10A cells), and the tumor cells are mouse breast cancer cells (4T1 cells) and human cervical cancer cells (HeLa cells).
从图10中可以看出，正常细胞(HEK-293T细胞，MCF-10A细胞)的细胞活性大约为90％，这表明本发明的纳米药物对正常细胞基本没有毒性。然而，肿瘤细胞的细胞活性均小于80％，这表明本发明的纳米药物试剂对肿瘤细胞有毒性。进一步的，从图8中可以看出，肿瘤细胞的细胞活性随着纳米药物剂浓度的增大而减小，随着时间的增加细胞活性也逐渐下降。由此可见，本发明的纳米药物有缓释的作用，对肿瘤细胞有抑制和杀死作用。It can be seen from Figure 10 that the cell viability of normal cells (HEK-293T cells, MCF-10A cells) is about 90%, which shows that the nanomedicine of the present invention has basically no toxicity to normal cells. However, the cell viability of the tumor cells was all less than 80%, which indicated that the nanomedicine agent of the present invention was toxic to tumor cells. Further, it can be seen from Figure 8 that the cell viability of the tumor cells decreases with the increase of the concentration of the nano-drug agent, and the cell viability gradually decreases with the increase of time. It can be seen that the nano-medicine of the present invention has the effect of sustained release, and has the effect of inhibiting and killing tumor cells.
图11-12展示了从细胞能量代谢的角度研究了实施例1制得的肿瘤靶向的纳米药物对癌症选择性机。从图9中看出,MSN载体对HeLa和MCF-10A细胞的ATP水平几乎没有影响,而FeCO-TPP、FeCO-TPP@MSN和FeCO-TPP@MSN@HA都随着线粒体量的减少而显著抑制了HeLa细胞的ATP水平,进而产生抗癌结果。从图10可以得到尽管FeCO-TPP和FeCO-TPP@MSN也抑制了MCF-10A细胞的ATP水平,但线粒体的相对数量并没有减少,可能是由于被释放的CO的保护作用造成的。由于CD44的识别能力，MCF-10A细胞的ATP水平和线粒体的相对数量反映了纳米颗粒对正常细胞的低细胞毒性。此外,正常细胞的基础呼吸的情况与ATP生产相似,最大呼吸和质子泄漏仍然保持不变,进一步表明了保护作用影响。Figures 11-12 show the study of the selectivity mechanism of the tumor-targeted nanomedicine prepared in Example 1 on cancer from the perspective of cell energy metabolism. It can be seen from Fig. 9 that the MSN carrier had almost no effect on the ATP level of HeLa and MCF-10A cells, while FeCO-TPP, FeCO-TPP@MSN and FeCO-TPP@MSN@HA all significantly decreased the amount of mitochondria Inhibited the ATP level of HeLa cells, which in turn produced anti-cancer results. It can be seen from Figure 10 that although FeCO-TPP and FeCO-TPP@MSN also inhibited the ATP level of MCF-10A cells, the relative number of mitochondria did not decrease, which may be caused by the protective effect of released CO. Due to the recognition ability of CD44, the ATP level and the relative number of mitochondria in MCF-10A cells reflected the low cytotoxicity of nanoparticles to normal cells. In addition, basal respiration in normal cells was similar to ATP production, and maximal respiration and proton leakage remained unchanged, further suggesting a protective effect.
图13展示了实施例1制得的肿瘤靶向的纳米药物进入小鼠体内的示踪图。采用4T1的肿瘤小鼠模型,采用FeCO-TPP@MSN@HA纳米药物进行小鼠体内尾静脉注射。从图11中的体内荧光成像追踪结果可以发现,在4T1肿瘤小鼠体内注射后,RITC标记的纳米颗粒逐渐积累到肿瘤中。注射2小时后解剖器官，荧光成像更清楚地表明被动和主动靶向能力，纳米颗粒主要分布在肝脏和肿瘤组织。FIG. 13 shows the trace diagram of the tumor-targeted nanomedicine prepared in Example 1 entering the mouse body. Using the 4T1 tumor mouse model, FeCO-TPP@MSN@HA nanomedicine was used for tail vein injection in mice. From the in vivo fluorescence imaging tracking results in Figure 11, it can be found that after in vivo injection in 4T1 tumor mice, RITC-labeled nanoparticles gradually accumulated into the tumor. Organs were dissected 2 hours after injection, and fluorescence imaging showed more clearly the passive and active targeting capabilities, and the nanoparticles were mainly distributed in the liver and tumor tissues.
图14展示了实施例1制得的肿瘤靶向的纳米药物在小鼠水平上的治疗效果的评价图。其中，图式(14a)表示为小鼠经过不同模式治疗20天内肿瘤体积的变化曲线，图示(14c)表示为小鼠经过不同模式治疗20天内小鼠体重的变化曲线。所述不同的治疗模式包括：模式一注射PSB缓冲溶液(对照组)，模式二注射MSN纳米颗粒(对照组)，模式三注射FeCO-TPP前药，模式四注射FeCO-TPP@MSN，模式五注射FeCO-TPP@MSN@HA。将PBS、实施例1制备的MSN，FeCO-TPP前药，FeCO-TPP@MSN，FeCO-TPP@MSN@HA灭菌处理，再将浓度为2mg/mL的不同组药剂分别通过尾静脉注入肿瘤小鼠体内，连续培养20天后，观测肿瘤小鼠肿瘤体积和小鼠体重的变化。Fig. 14 shows the evaluation diagram of the therapeutic effect of the tumor-targeted nanomedicine prepared in Example 1 at the level of mice. Among them, the diagram (14a) represents the change curve of the tumor volume of the mice treated with different modes within 20 days, and the diagram (14c) represents the change curve of the mouse body weight within 20 days of the mice treated with different modes. The different treatment modes include: mode one injection of PSB buffer solution (control group), mode two injection of MSN nanoparticles (control group), mode three injection of FeCO-TPP prodrug, mode four injection of FeCO-TPP@MSN, mode five FeCO-TPP@MSN@HA was injected. Sterilize PBS, MSN prepared in Example 1, FeCO-TPP prodrug, FeCO-TPP@MSN, FeCO-TPP@MSN@HA, and then inject different groups of agents with a concentration of 2mg/mL into the tumor through the tail vein respectively In mice, after continuous culture for 20 days, the changes of tumor volume and body weight of tumor mice were observed.
从图14中可以看出，注射实施例1制备的FeCO-TPP@MSN@HA的小鼠的体重无明显变化，这表明本发明的纳米药物对小鼠没明显的毒副作用。此外，注射实施例1制备的FeCO-TPP@MSN@HA的肿瘤体积无明显增加，而注入PBS和MSN后小鼠的肿瘤体积显著增加，这表明本发明的纳米药物对肿瘤具有明显的抑制效果。It can be seen from Figure 14 that the body weight of the mice injected with FeCO-TPP@MSN@HA prepared in Example 1 did not change significantly, which indicates that the nanomedicine of the present invention has no obvious toxic and side effects on mice. In addition, the tumor volume of the injected FeCO-TPP@MSN@HA prepared in Example 1 did not increase significantly, while the tumor volume of mice injected with PBS and MSN increased significantly, which indicated that the nanomedicine of the present invention has a significant inhibitory effect on tumors .
图15展示了小鼠经过不同模式治疗20天后的主要的器官(心、肝、脾、肺、肾)以及肿瘤组织的生理组织切片图。将PBS、实施例1制备的MSN，CO前药，FeCO-TPP@MSN，FeCO-TPP@MSN@HA灭菌处理，再将浓度为1mg/mL的不同组药剂分别通过尾静脉注入荷瘤小鼠的4T1肿瘤内部，经过20天培养后，观测小鼠的主要器官(心、肝、脾、肺、肾)和肿瘤的组织切片。从切片测试结果中可以看出，注射实施例1制得的纳米药物有效的杀死了肿瘤细胞，并没有对小鼠的主要器官(心、肝、脾、肺、肾)造成明显损伤，这表明本发明的纳米药物对正常组织没有造成明显的毒副作用，而对肿瘤细胞有明显的杀伤作用。Figure 15 shows the physiological tissue slices of the main organs (heart, liver, spleen, lung, kidney) and tumor tissues of the mice treated with different modes for 20 days. Sterilize PBS, MSN prepared in Example 1, CO prodrug, FeCO-TPP@MSN, FeCO-TPP@MSN@HA, and then inject different groups of agents with a concentration of 1 mg/mL into the tumor-bearing tumor through the tail vein respectively. Inside the 4T1 tumor of the mouse, after 20 days of culture, the main organs (heart, liver, spleen, lung, kidney) and tumor tissue sections of the mouse were observed. As can be seen from the slice test results, the injection of the nano-medicine prepared in Example 1 effectively killed the tumor cells, and did not cause significant damage to the main organs (heart, liver, spleen, lung, kidney) of the mouse. It shows that the nano-medicine of the present invention does not cause obvious toxic and side effects to normal tissues, but has obvious killing effect on tumor cells.
本发明成功设计并制备一种具有肿瘤细胞线粒体靶向传输、可控CO释放功能的新型智能纳米药物FeCO-TPP@MSN@HA,带负电的MSN载体高效装载带正电的CO前药，然后包裹HA靶向分子，构建集多级靶向和多级控释功能于一体的智能纳米药物。通过EPR效应，实现FeCO-TPP@MSN@HA被动靶向进入肿瘤组织(第一级靶向)，通过HA分子对CD44蛋白的靶向识别作用，实现对过度表达CD44蛋白的肿瘤细胞的靶向药物传输(第二级靶向)；通过正电性CO前药与酸性环境的质子交换，实现酸响应性CO前药控释(第一级控释)；释放出来的CO前药靶向摄入细胞线粒体中(第三级靶向)；CO前药在线粒体内ROS的作用下响应性控释CO(第二级控释)。最后，本发明的纳米气体药物能够实现针对恶性肿瘤的响应性释放和无创气体治疗控，且具有高效低毒的治疗效果。The present invention successfully designs and prepares a new type of intelligent nano-drug FeCO-TPP@MSN@HA, which has the functions of tumor cell mitochondria targeted delivery and controllable CO release. The negatively charged MSN carrier efficiently loads the positively charged CO prodrug, and then Encapsulate HA targeting molecules to construct smart nano-drugs that integrate multi-level targeting and multi-level controlled release functions. Through the EPR effect, the passive targeting of FeCO-TPP@MSN@HA into tumor tissue (first-level targeting) is achieved, and the targeting of tumor cells overexpressing CD44 protein is achieved through the targeted recognition of CD44 protein by HA molecules Drug delivery (second-level targeting); acid-responsive CO prodrug controlled release (first-level controlled release) through proton exchange between positively charged CO prodrug and acidic environment; released CO prodrug targeted uptake Into the mitochondria of cells (third-level targeting); CO prodrugs respond to controlled release of CO under the action of ROS in mitochondria (second-level controlled release). Finally, the nano gas drug of the present invention can realize responsive release and non-invasive gas treatment control for malignant tumors, and has high efficiency and low toxicity.
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，以上实施方式仅是用于解释权利要求书。然本发明的保护范围并不局限于说明书。任何熟悉本技术领域的技术人员在本发明披露的技术范围内，可轻易想到的变化或者替换，都包含在本发明的保护范围之内。The above-mentioned embodiment is a preferred implementation mode of the present invention, but the implementation mode of the present invention is not limited by the above-mentioned embodiment, and the above-mentioned implementation mode is only for explaining the claims. However, the protection scope of the present invention is not limited to the description. Any changes or substitutions that can be easily conceived by any person skilled in the art within the technical scope disclosed in the present invention are included in the protection scope of the present invention.
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| CN201910967255.8ACN110538330B (en) | 2019-10-12 | 2019-10-12 | Medicine for mitochondria targeted CO transmission and preparation method thereof |
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| CN201910967255.8ACN110538330B (en) | 2019-10-12 | 2019-10-12 | Medicine for mitochondria targeted CO transmission and preparation method thereof |
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