CN115779081A - A lipid composite nanocarrier co-carrying siSkp2 and verteporfin and its preparation method and application - Google Patents
A lipid composite nanocarrier co-carrying siSkp2 and verteporfin and its preparation method and applicationDownload PDFInfo
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Abstract
Translated fromChinese
Description
Translated fromChinese技术领域technical field
本发明属于纳米药物领域，具体地涉及一种共载siSkp2与维替泊芬的脂质复合纳米载体及其制备方法与应用。The invention belongs to the field of nanomedicine, and in particular relates to a lipid composite nanocarrier co-carrying siSkp2 and verteporfin, a preparation method and application thereof.
背景技术Background technique
当今癌症以其高致死率、治疗周期长且易复发转移的特征，已成为威胁人类生命安全的顽疾之一。前列腺癌的发病率逐年升高，在男性泌尿系统中已经成为发病率最高的肿瘤。目前对于前列腺癌的治疗大部分停留在手术化疗等层面，这些方法愈后效果较差、副作用较大，有可能将会造成患者的二次伤害。目前，纳米药物的迅速发展给众多癌症患者带来了福音，其治疗效果好、靶向性高且毒副作用小，已成为治疗癌症的首选方法。而通过赋予纳米药物以各种治疗机制联合抑制肿瘤的发展已成为研究热点。Nowadays, cancer has become one of the persistent diseases threatening human life safety due to its high mortality rate, long treatment cycle and easy recurrence and metastasis. The incidence of prostate cancer is increasing year by year, and it has become the tumor with the highest incidence in male urinary system. At present, most of the treatments for prostate cancer remain at the level of surgery and chemotherapy. These methods have poor curative effects and relatively large side effects, which may cause secondary harm to patients. At present, the rapid development of nanomedicine has brought good news to many cancer patients. It has good therapeutic effect, high targeting and low side effects, and has become the first choice for cancer treatment. The development of combined tumor suppression by endowing nanomedicines with various therapeutic mechanisms has become a research hotspot.
小干扰RNA(small interfering RNA，siRNA)作为RNA干扰(RNA interference，RNAi)技术的重要效应分子，能有效降解基因转录后的mRNA，抑制相应蛋白合成，实现基因沉默效应。siRNA药物具有可设计靶向、瞬时沉默、合成方便、靶点特异性强等优势，在肿瘤治疗等方面被广泛应用。但作为负电性的生物大分子，裸siRNA在进入人体后存在易被核酸酶降解、肾小球滤过和半衰期较短的问题，导致siRNA药物在临床上的应用受到了极大的限制约束。因此，如何选择合适的载体构建高效安全的体内递送系统以提高siRNA在人体内的稳定性与靶细胞中的基因沉默效率，是RNAi技术实现临床转化的关键。As an important effector molecule of RNA interference (RNAi) technology, small interfering RNA (small interfering RNA, siRNA) can effectively degrade mRNA after gene transcription, inhibit corresponding protein synthesis, and realize gene silencing effect. siRNA drugs have the advantages of designable targeting, transient silencing, convenient synthesis, and strong target specificity, and are widely used in tumor therapy and other aspects. However, as a negatively charged biomacromolecule, naked siRNA has the problems of being easily degraded by nucleases, glomerular filtration, and short half-life after entering the human body, which has greatly restricted the clinical application of siRNA drugs. Therefore, how to select a suitable carrier to construct an efficient and safe in vivo delivery system to improve the stability of siRNA in the human body and the gene silencing efficiency in target cells is the key to the clinical transformation of RNAi technology.
本研究利用Cy3-siRNA与卟啉化合物维替泊芬(VP)先组装形成纳米递送系统，再利用脂质体进行包裹，构建一种新的脂质纳米药物(Lipo@Cy3-siSkp2/VP NPs)。所设计的纳米药物中的VP具有光敏性质，在光照下，可以产生单线态氧，杀伤肿瘤。所设计的纳米药物中的siRNA可以靶向敲低Skp2蛋白，发挥肿瘤抑制作用。光动力治疗与RNAi技术的联合，进一步提升该系统对于肿瘤的抑制效果，真正实现“1+1>2”的效果。基于脂质体具有水分散性好、生物相容性强、生物利用度高和生物可降解性等特点，在已形成的纳米递送系统上包覆脂质体，进一步提高递送系统的稳定性。In this study, Cy3-siRNA and the porphyrin compound verteporfin (VP) were first assembled to form a nano-delivery system, and then encapsulated by liposomes to construct a new lipid nano-drug (Lipo@Cy3-siSkp2/VP NPs ). The VP in the designed nanomedicine has photosensitivity, under the light, it can generate singlet oxygen and kill the tumor. The siRNA in the designed nanomedicine can target and knock down the Skp2 protein and play a tumor suppressive role. The combination of photodynamic therapy and RNAi technology further enhances the system's inhibitory effect on tumors and truly achieves the effect of "1+1>2". Based on the characteristics of liposomes such as good water dispersibility, strong biocompatibility, high bioavailability and biodegradability, liposomes are coated on the formed nano-delivery system to further improve the stability of the delivery system.
发明内容Contents of the invention
本发明的目的是提供一种共载siSkp2与维替泊芬的脂质复合纳米载体及其制备方法与应用。The object of the present invention is to provide a lipid composite nanocarrier co-carrying siSkp2 and verteporfin and its preparation method and application.
本发明利用Cy3-siSkp2与VP之间的相互作用自组装形成纳米粒子(Cy3-siRNA/VPNPs)，并利用脂质体(Liposome，Lipo)对Cy3-siSkp2/VPNPs进行包裹，构建脂质纳米共递送递送系统(Lipo@Cy3-siSkp2/VPNPs)。本发明可将siRNA和维替泊芬同时携带进入前列腺肿瘤细胞，二者发挥基因治疗与光动力治疗效果，协同抑制前列腺癌细胞增殖。The present invention uses the interaction between Cy3-siSkp2 and VP to self-assemble to form nanoparticles (Cy3-siRNA/VPNPs), and uses liposomes (Liposome, Lipo) to wrap Cy3-siSkp2/VPNPs to construct lipid nanoco- Delivery delivery system (Lipo@Cy3-siSkp2/VPNPs). The present invention can simultaneously carry siRNA and verteporfin into prostate tumor cells, and the two exert gene therapy and photodynamic therapy effects to synergistically inhibit the proliferation of prostate cancer cells.
为实现上述目的，本发明采用如下技术方案：To achieve the above object, the present invention adopts the following technical solutions:
本发明首先提供了一种共载siSkp2与维替泊芬的脂质复合纳米载体，包含维替泊芬、skp2沉默性siRNA和阳离子脂质体。The present invention firstly provides a lipid composite nanocarrier co-carrying siSkp2 and verteporfin, comprising verteporfin, skp2 silencing siRNA and cationic liposome.
进一步地，所述siRNA的序列：sense5'-3'：GGAGUGACAAAGACUUUGU，antisense5'-3'：ACAAAGUCUUUGUCACUCC(现有公开序列)。Further, the sequence of the siRNA: sense5'-3': GGAGUGACAAAGACUUUGU, antisense5'-3': ACAAAGUCUUUGUCACUCC (current public sequence).
进一步地，所述阳离子脂质体包括DMPC、DSPE-mPEG2000和DOTAP。Further, the cationic liposome includes DMPC, DSPE-mPEG2000 and DOTAP.
更进一步地，所述脂质体中DMPC、DSPE-mPEG2000和DOTAP的质量比为2-5：0.1-0.3：1。Furthermore, the mass ratio of DMPC, DSPE-mPEG2000 and DOTAP in the liposome is 2-5:0.1-0.3:1.
更进一步地，所述脂质体的制备方法如下：Further, the preparation method of the liposome is as follows:
DMPC、DSPE-mPEG2000和DOTAP用有机溶剂溶解混合，减压除去有机溶剂，再加入DEPC水水合分散，所得溶液采用挤出器制备脂质体，并控制粒径小于200nm。DMPC, DSPE-mPEG2000 and DOTAP were dissolved and mixed with an organic solvent, the organic solvent was removed under reduced pressure, and then DEPC water was added to hydrate and disperse. The resulting solution was used to prepare liposomes with an extruder, and the particle size was controlled to be less than 200nm.
进一步地，所述siRNA的终浓度为2-8μM，所述维替泊芬的终浓度为100-200μM。Further, the final concentration of the siRNA is 2-8 μM, and the final concentration of the verteporfin is 100-200 μM.
本发明还提供了所述脂质复合纳米载体的制备方法，具体包括以下步骤：The present invention also provides a preparation method of the lipid composite nanocarrier, specifically comprising the following steps:
(1)在无菌操作台中将siRNA干粉溶于DEPC处理水，制备得到200μM siRNA母液；将VP粉末在DMSO里充分溶解，得5mM VP母液。取30μL，5mM VP母液与25μL，200μM siRNA溶液充分混匀，加入45μLDEPC水，于室温，80rpm条件下的摇床避光孵育30min，即得100μL siSkp2/VP纳米药物(Cy3-siRNA/VP NPs＝50μM/1500μM)，于4℃条件下避光保存。(1) Dissolve siRNA dry powder in DEPC-treated water in a sterile operating bench to prepare 200 μM siRNA stock solution; fully dissolve VP powder in DMSO to obtain 5 mM VP stock solution. Take 30 μL, 5 mM VP stock solution and 25 μL, 200 μM siRNA solution, mix thoroughly, add 45 μL DEPC water, and incubate at room temperature, 80 rpm, in the dark for 30 min on a shaking table to obtain 100 μL siSkp2/VP nanomedicine (Cy3-siRNA/VP NPs= 50μM/1500μM), stored at 4°C in the dark.
(2)取3.7mg DMPC、0.23mg DSPE-mPEG2000、1mg DOTAP，分别使用氯仿(分析纯)溶解得到浓度为10mg/ml的母液后混合，后加入氯仿(分析纯)补齐至1mL；利用旋蒸仪蒸发有机溶剂；旋蒸结束后使用氮气干燥20min；干燥结束后加入1640μL的DEPC水于45℃，常压条件下水合1h制备得到浓度为3mg/mL的脂质。利用配备200nm膜的脂质体挤出器对3mg/mL的脂质挤出来回21次得到脂质体，记为Liposome(Lipo)。(2) Take 3.7mg of DMPC, 0.23mg of DSPE-mPEG2000, and 1mg of DOTAP, and dissolve them in chloroform (analytical pure) to obtain a mother liquor with a concentration of 10mg/ml, mix them, and then add chloroform (analytical pure) to make up to 1mL; Evaporate the organic solvent with an evaporator; dry with nitrogen gas for 20 min after rotary evaporation; add 1640 μL of DEPC water at 45°C after drying, and hydrate for 1 h under normal pressure to prepare a lipid with a concentration of 3 mg/mL. A liposome extruder equipped with a 200nm membrane was used to extrude 3 mg/mL lipid back and forth 21 times to obtain a liposome, which was designated as Liposome (Lipo).
(3)将Lipo(终浓度为1.5mg/mL)与Cy3-siRNA/VP NPs(Cy3-siRNA/VP终浓度为5μM/150μM)等体积混合后，使用配备100nm膜的脂质体挤出器共挤出，8000g离心10min，收集沉淀，即为纳米粒子Lipo@Cy3-siRNA/VP NPs，置于-80℃超低温冰箱中存储备用。(3) After mixing equal volumes of Lipo (final concentration 1.5mg/mL) and Cy3-siRNA/VP NPs (final concentration Cy3-siRNA/VP 5μM/150μM), use a liposome extruder equipped with a 100nm membrane Co-extrude, centrifuge at 8000g for 10min, collect the precipitate, which is the nanoparticle Lipo@Cy3-siRNA/VP NPs, and store it in a -80°C ultra-low temperature refrigerator for later use.
最后，本发明提供了所述脂质复合纳米载体在制备抗前列腺癌药物中的应用。Finally, the present invention provides the application of the lipid complex nanocarrier in the preparation of anti-prostate cancer drugs.
本发明的有益效果在于：本发明设计利用Cy3-siSkp2与VP之间的相互作用自组装形成纳米粒子(Cy3-siRNA/VPNPs)，并进一步利用脂质体(Liposome，Lipo)对Cy3-siSkp2/VPNPs进行包裹，构建脂质纳米共递送递送系统(Lipo@Cy3-siSkp2/VPNPs)。本发明可保护siRNA不被血清降解，提高siRNA在人体内的稳定性，并具有光刺激响应性质，可高效地将siRNA和维替泊芬同时携带进入前列腺肿瘤细胞，二者发挥双重基因治疗与光动力治疗效果，协同抑制前列腺癌细胞增殖。The beneficial effect of the present invention is: the present invention design utilizes the interaction self-assembly between Cy3-siSkp2 and VP to form nanoparticles (Cy3-siRNA/VPNPs), and further utilize liposome (Liposome, Lipo) to Cy3-siSkp2/ VPNPs were packaged to construct a lipid nano-co-delivery delivery system (Lipo@Cy3-siSkp2/VPNPs). The invention can protect siRNA from being degraded by serum, improve the stability of siRNA in the human body, and has light-stimulus response properties, and can efficiently carry siRNA and verteporfin into prostate tumor cells at the same time. Photodynamic therapy effect, synergistic inhibition of prostate cancer cell proliferation.
附图说明Description of drawings
下面参照附图结合实施例对本发明作进一步的说明。The present invention will be further described below in conjunction with the embodiments with reference to the accompanying drawings.
图1为Cy3-siRNA/VP NPs、Lipo和Lipo@Cy3-siRNA/VP NPs的粒径。Figure 1 shows the particle size of Cy3-siRNA/VP NPs, Lipo and Lipo@Cy3-siRNA/VP NPs.
图2为Cy3-siRNA、VP、Cy3-siRNA/VP NPs、Lipo、Lipo@Cy3-siRNA/VP NPs的zeta电位；数据以平均值±标准差表示(n＝3)。Figure 2 shows the zeta potentials of Cy3-siRNA, VP, Cy3-siRNA/VP NPs, Lipo, Lipo@Cy3-siRNA/VP NPs; the data are expressed as mean ± SD (n=3).
图3为Cy3-siRNA/VP NPs、Lipo、Lipo@Cy3-siRNA/VP NPs的TEM表征；其中标尺为100nm。Figure 3 is the TEM characterization of Cy3-siRNA/VP NPs, Lipo, Lipo@Cy3-siRNA/VP NPs; where the scale bar is 100nm.
图4为利用CLSM表征Lipo@Cy3-siRNA/VP NPs的构建；其中标尺为1μm。Figure 4 is the construction of Lipo@Cy3-siRNA/VP NPs characterized by CLSM; the scale bar is 1 μm.
图5为利用荧光光谱仪探究siRNA/VP NPs、Lipo@Cy3-siRNA/VP NPs稳定性，数据以平均值±标准差表示(n＝3)。Figure 5 is the use of fluorescence spectrometer to explore the stability of siRNA/VP NPs and Lipo@Cy3-siRNA/VP NPs, and the data are represented by mean ± standard deviation (n=3).
图6中A表示不同浓度VP在光照后对前列腺肿瘤细胞生长的抑制效果考察，B表示Cy3-siRNA、VP、Cy3-siRNA/VP NPs和Lipo@Cy3-siRNA/VP NPs对前列腺肿瘤细胞生长的抑制效果考察；数据以平均值±标准差表示(n＝3)，显著性(*P<0.05，***P<0.001)。In Figure 6, A represents the investigation of the inhibitory effects of different concentrations of VP on the growth of prostate tumor cells after illumination, and B represents the effects of Cy3-siRNA, VP, Cy3-siRNA/VP NPs and Lipo@Cy3-siRNA/VP NPs on the growth of prostate tumor cells Inhibition effect inspection; data expressed as mean ± standard deviation (n=3), significant (* P<0.05,*** P<0.001).
具体实施方式Detailed ways
实施例1Cy3-siRNA/VP NPs、Lipo和Lipo@Cy3-siRNA/VP NPs的制备Preparation of Example 1Cy3-siRNA/VP NPs, Lipo and Lipo@Cy3-siRNA/VP NPs
(1)在无菌操作台中将siRNA干粉溶于DEPC处理水，制备得到200μM siRNA母液；将VP粉末在DMSO里充分溶解，得5mM VP母液。取30μL，5mM VP母液与25μL，200μM siRNA溶液充分混匀，加入45μLDEPC水，于室温，80rpm条件下的摇床避光孵育30min，即得100μL siSkp2/VP纳米药物(Cy3-siRNA/VP NPs＝50μM/1500μM)，于4℃条件下避光保存。(1) Dissolve siRNA dry powder in DEPC-treated water in a sterile operating bench to prepare 200 μM siRNA stock solution; fully dissolve VP powder in DMSO to obtain 5 mM VP stock solution. Take 30 μL, 5 mM VP stock solution and 25 μL, 200 μM siRNA solution, mix thoroughly, add 45 μL DEPC water, and incubate at room temperature, 80 rpm, in the dark for 30 min on a shaking table to obtain 100 μL siSkp2/VP nanomedicine (Cy3-siRNA/VP NPs= 50μM/1500μM), stored at 4°C in the dark.
(2)取3.7mg DMPC、0.23mg DSPE-mPEG2000、1mg DOTAP，分别使用氯仿(分析纯)溶解得到浓度为10mg/ml的母液后混合，后加入氯仿(分析纯)补齐至1mL；利用旋蒸仪蒸发有机溶剂；旋蒸结束后使用氮气干燥20min；干燥结束后加入1640μL的DEPC水于45℃，常压条件下水合1h制备得到浓度为3mg/mL的脂质。利用配备200nm膜的脂质体挤出器对3mg/mL的脂质挤出来回21次得到脂质体，记为Liposome(Lipo)。(2) Take 3.7mg of DMPC, 0.23mg of DSPE-mPEG2000, and 1mg of DOTAP, and dissolve them in chloroform (analytical pure) to obtain a mother liquor with a concentration of 10mg/ml, mix them, and then add chloroform (analytical pure) to make up to 1mL; Evaporate the organic solvent with an evaporator; dry with nitrogen gas for 20 min after rotary evaporation; add 1640 μL of DEPC water at 45°C after drying, and hydrate for 1 h under normal pressure to prepare a lipid with a concentration of 3 mg/mL. A liposome extruder equipped with a 200nm membrane was used to extrude 3 mg/mL lipid back and forth 21 times to obtain a liposome, which was designated as Liposome (Lipo).
(3)将Lipo(终浓度为1.5mg/mL)与Cy3-siRNA/VP NPs(Cy3-siRNA/VP终浓度为5μM/150μM)等体积混合后，使用配备100nm膜的脂质体挤出器共挤出，8000g离心10min，收集沉淀，即为纳米粒子Lipo@Cy3-siRNA/VP NPs，置于-80℃超低温冰箱中存储备用。(3) After mixing equal volumes of Lipo (final concentration 1.5mg/mL) and Cy3-siRNA/VP NPs (final concentration Cy3-siRNA/VP 5μM/150μM), use a liposome extruder equipped with a 100nm membrane Co-extrude, centrifuge at 8000g for 10min, collect the precipitate, which is the nanoparticle Lipo@Cy3-siRNA/VP NPs, and store it in a -80°C ultra-low temperature refrigerator for later use.
实施例2DLS测定纳米粒子的粒径分布和表面电位Embodiment 2DLS measures the size distribution and surface potential of nanoparticles
利用动态光散射仪(DLS)对Cy3-siRNA/VP NPs、Lipo和Lipo@Cy3-siRNA/VP NPs的粒径分布和表面电位的分布进行测量表征。每个样品需要用DEPC处理水进行稀释，并重复测试三次，温度设置为25℃。The particle size distribution and surface potential distribution of Cy3-siRNA/VP NPs, Lipo and Lipo@Cy3-siRNA/VP NPs were measured and characterized by dynamic light scattering (DLS). Each sample needs to be diluted with DEPC-treated water, and the test is repeated three times, and the temperature is set at 25 °C.
实验结果：如图1所示，通过马尔文粒度分析仪的动态光散射法(DLS)对Cy3-siRNA/VP NPs、Lipo和Lipo@Cy3-siRNA/VP NPs的粒径分布进行表征。三种粒子的粒径均呈单峰分布。其中，Cy3-siRNA/VP NPs的平均粒径为91nm；Lipo的平均粒径为164nm；Lipo@Cy3-siRNA/VP NPs的平均粒径为255nm。该结果初步验证，Lipo@Cy3-siRNA/VP NPs成功构建。结果如图2所示，游离的Cy3-siRNA的平均电位为-11mV，游离的VP的平均电位为11.7mV，纳米粒子Cy3-siRNA/VP NPs的平均电位为8.4mV，Lipo的平均电位为20.4mV，包覆后的Lipo@Cy3-siRNA/VP NPs平均电位为30.6mv，与Cy3-siRNA/VP NPs相比，Lipo@Cy3-siRNA/VP NPs的电位值正移，可以初步说明带正电的脂质体已成功包覆于Cy3-siRNA/VP NPs表面。Experimental results: As shown in Figure 1, the particle size distribution of Cy3-siRNA/VP NPs, Lipo and Lipo@Cy3-siRNA/VP NPs were characterized by the dynamic light scattering method (DLS) of the Malvern particle size analyzer. The particle sizes of the three kinds of particles all showed a unimodal distribution. Among them, the average particle size of Cy3-siRNA/VP NPs is 91nm; the average particle size of Lipo is 164nm; the average particle size of Lipo@Cy3-siRNA/VP NPs is 255nm. The result was preliminarily verified that Lipo@Cy3-siRNA/VP NPs were successfully constructed. The results are shown in Figure 2, the average potential of free Cy3-siRNA is -11mV, the average potential of free VP is 11.7mV, the average potential of nanoparticles Cy3-siRNA/VP NPs is 8.4mV, and the average potential of Lipo is 20.4 mV, the average potential of Lipo@Cy3-siRNA/VP NPs after coating is 30.6mv. Compared with Cy3-siRNA/VP NPs, the potential value of Lipo@Cy3-siRNA/VP NPs shifted positively, which can preliminarily indicate that the positively charged liposomes have been successfully coated on the surface of Cy3-siRNA/VP NPs.
实施例3TEM表征光照前后纳米药物的形貌
使用透射电子显微镜(TEM)观察690nm光照前后Cy3-siRNA/VP NPs、Lipo和Lipo@Cy3-siRNA/VP NPs的形貌和尺寸，将上述纳米粒子与磷钨酸等体积混合滴加在铜网上，等待1h后吸去上清，待铜网干燥，用透射电子显微镜进行测试，测试加速电压为200KV。Use a transmission electron microscope (TEM) to observe the morphology and size of Cy3-siRNA/VP NPs, Lipo and Lipo@Cy3-siRNA/VP NPs before and after 690nm illumination, and mix the above-mentioned nanoparticles with equal volumes of phosphotungstic acid and drop them on the copper grid , wait for 1 hour, suck off the supernatant, wait for the copper grid to dry, and test it with a transmission electron microscope. The test acceleration voltage is 200KV.
实验结果：如图3所示，可以清晰地观察到Cy3-siRNA/VP NPs是粒径约为90nm的实心球形颗粒；Lipo是粒径约为120nm的空心球形囊泡；Lipo@Cy3-siRNA/VP NPs可见膜包裹实心球状粒子的核壳结构，其粒径约为200nm，进一步说明基于脂质体的纳米递送系统构建成功。光照后，Lipo并不会受到光照的影响故保持着原来的形态。而Cy3-siRNA/VP NPs和Lipo@Cy3-siRNA/VP NPs组，可见原有圆形形态改变。该现象说明Cy3-siRNA/VP NPs和Lipo@Cy3-siRNA/VP NPs具有光响应解离性质，由于其中VP的存在，在光照刺激后产生单线态氧，破坏Cy3-siRNA/VP NPs和Lipo@Cy3-siRNA/VP NPs的结构，导致其发生解离，因此原有的颗粒形态发生改变。Experimental results: As shown in Figure 3, it can be clearly observed that Cy3-siRNA/VP NPs are solid spherical particles with a particle size of about 90 nm; Lipo is a hollow spherical vesicle with a particle size of about 120 nm; Lipo@Cy3-siRNA/ The core-shell structure of solid spherical particles surrounded by membranes can be seen in VP NPs, and its particle size is about 200nm, further indicating that the liposome-based nano-delivery system was successfully constructed. After lighting, Lipo will not be affected by the light so it maintains its original shape. In the Cy3-siRNA/VP NPs and Lipo@Cy3-siRNA/VP NPs groups, changes in the original circular shape can be seen. This phenomenon shows that Cy3-siRNA/VP NPs and Lipo@Cy3-siRNA/VP NPs have light-responsive dissociation properties. Due to the existence of VP, singlet oxygen is generated after light stimulation, which destroys Cy3-siRNA/VP NPs and Lipo@ The structure of Cy3-siRNA/VP NPs leads to its dissociation, so the original particle morphology changes.
实施例4激光共聚焦显微镜表征纳米递送系统的构建Example 4 Confocal laser microscopy characterization of the construction of nano-delivery systems
将Lipo@Cy3-siRNA/VP NPs重悬在1mLPBS中，其中含有1μL DiO细胞膜染料，37℃下孵育10min。在4℃下离心8000g，10min，去上清，沉淀用50μL重悬，浓缩至终浓度Cy3-siRNA/VP＝40μM/800μM。取10μL Lipo@Cy3-siRNA/VP NPs滴在共聚焦皿中间，盖上盖玻片，静止3min，共聚焦拍摄。Resuspend Lipo@Cy3-siRNA/VP NPs in 1 mL of LPBS containing 1 μL of DiO cell membrane dye, and incubate at 37°C for 10 min. Centrifuge at 8000 g for 10 min at 4°C, remove the supernatant, resuspend the pellet in 50 μL, and concentrate to a final concentration of Cy3-siRNA/VP=40 μM/800 μM. Take 10 μL of Lipo@Cy3-siRNA/VP NPs and drop in the middle of the confocal dish, cover with a cover glass, let it rest for 3 minutes, and take confocal photography.
实验结果：我们通过激光扫描共聚焦显微镜(Confocal laser scanningmicroscope，CLSM)观察Lipo@Cy3-siRNA/VP NPs的构建情况。Cy3-siRNA的激发波长为550nm，发射波长为570nm，颜色标记为绿色；VP的激发波长为550nm，发射波长为690nm，颜色标记为红色；利用CellMask染料对脂质体进行染色，其激发波长为522nm，发射波长为535nm，颜色标记为紫色。如图4所示，我们可以看到Lipo、Cy3-siRNA与VP的荧光信号在同一位置上发生重叠，由此可以进一步证实我们已将Lipo成功包覆于Cy3-siRNA/VP NPs，Lipo@Cy3-siRNA/VP NPs构建成功。Experimental results: We observed the construction of Lipo@Cy3-siRNA/VP NPs by Confocal laser scanning microscope (CLSM). The excitation wavelength of Cy3-siRNA is 550nm, the emission wavelength is 570nm, and the color is marked as green; the excitation wavelength of VP is 550nm, the emission wavelength is 690nm, and the color is marked as red; CellMask dyes are used to stain liposomes, and the excitation wavelength is 522nm, the emission wavelength is 535nm, and the color is marked purple. As shown in Figure 4, we can see that the fluorescence signals of Lipo, Cy3-siRNA and VP overlap at the same position, which further confirms that we have successfully coated Lipo on Cy3-siRNA/VP NPs, Lipo@Cy3 -siRNA/VP NPs were constructed successfully.
实施例5Lipo@Cy3-siRNA/VP NPs稳定性考察Example 5 Lipo@Cy3-siRNA/VP NPs Stability Investigation
(1)制作Cy3-siRNA和VP的荧光标曲：配制不同梯度的Cy3-siRNA和VP溶液，Cy3-siRNA设置浓度为0、0.05、0.1、0.15、0.2、0.25、0.3μM，VP设置浓度为0、1、2、4、6、8、10μM。然后，用安捷伦荧光光谱仪检测相应的荧光强度，拟合成标准曲线。(1) Make the fluorescence standard of Cy3-siRNA and VP: prepare different gradients of Cy3-siRNA and VP solutions, set the concentration of Cy3-siRNA as 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3μM, and set the concentration of VP as 0, 1, 2, 4, 6, 8, 10 μM. Then, the corresponding fluorescence intensity was detected with an Agilent fluorescence spectrometer, and a standard curve was fitted.
(2)分别在0h、1h、3h、6h、9h、12h各时间点向各EP管中加入2μLCy3-siRNA/VP NPs或Lipo@Cy3-siRNA/VP NPs(Cy3-siRNA/VP终浓度为0.50/15μM)+198μLPBS(pH＝4.0/7.5/10％FBS)，并做好标记；(2) Add 2 μL of Cy3-siRNA/VP NPs or Lipo@Cy3-siRNA/VP NPs to each EP tube at each time point of 0h, 1h, 3h, 6h, 9h, and 12h (the final concentration of Cy3-siRNA/VP is 0.50 /15μM)+198μL PBS (pH=4.0/7.5/10%FBS), and marked;
(3)将各样品在8000g的条件下离心10min，收集上清并使用200μL的DEPC处理水重悬沉淀；(3) Centrifuge each sample at 8000g for 10min, collect the supernatant and resuspend the pellet with 200μL of DEPC-treated water;
(4)使用安捷伦荧光光谱仪测定上清液与沉淀重悬液的荧光强度，并带入标曲计算Cy3-siRNA和VP浓度；(4) Use an Agilent fluorescence spectrometer to measure the fluorescence intensity of the supernatant and the precipitate resuspension, and bring it into the bracket to calculate the concentration of Cy3-siRNA and VP;
实验结果：我们通过检测Cy3-siRNA/VP NPs与Lipo@Cy3-siRNA/VP NPs分别于PBS(pH＝5)、PBS(pH＝7.5)和PBS(pH＝7.5)+10％FBS环境下，孵育不同时间点(0、1、3、6、9、12h)，检测上清和沉淀的荧光强度，从而得到相对应的siRNA释放率，以考察Cy3-siRNA/VPNPs和Lipo@Cy3-siRNA/VP NPs的稳定性。结果如图5所示，Cy3-siRNA/VP NPs粒子在pH＝7.5的PBS溶液中较为稳定，pH＝5的PBS溶液中次之，在含10％FBS的pH＝7.5PBS溶液中最不稳定，当孵育时间为6h，siRNA释放率就高达95％。而与Cy3-siRNA/VP NPs相比，Lipo@Cy3-siRNA/VP NPs在各溶液中的siRNA释放率均有一定程度的下降，其中，在含10％FBS的pH＝7.5 PBS溶液中，孵育时间为6h时，siRNA的释放率<50％。以上结果说明脂质体包覆后得到的Lipo@Cy3-siRNA/VP NPs可以有效地提高Cy3-siRNA/VP NPs的稳定性，尤其是对其在血清中稳定性的提高，有利于其更好地在人体内发挥效用。Experimental results: We tested Cy3-siRNA/VP NPs and Lipo@Cy3-siRNA/VP NPs in PBS (pH=5), PBS (pH=7.5) and PBS (pH=7.5)+10% FBS respectively, Incubate at different time points (0, 1, 3, 6, 9, 12h), detect the fluorescence intensity of the supernatant and the precipitate, so as to obtain the corresponding siRNA release rate, to investigate Cy3-siRNA/VPNPs and Lipo@Cy3-siRNA/VP Stability of NPs. The results are shown in Figure 5, Cy3-siRNA/VP NPs particles are relatively stable in the PBS solution of pH=7.5, followed by the PBS solution of pH=5, and the most unstable in the PBS solution of pH=7.5 containing 10% FBS , when the incubation time is 6h, the release rate of siRNA is as high as 95%. Compared with Cy3-siRNA/VP NPs, the siRNA release rate of Lipo@Cy3-siRNA/VP NPs in each solution decreased to a certain extent. Among them, in the pH=7.5 PBS solution containing 10% FBS, incubation When the time is 6h, the release rate of siRNA is <50%. The above results show that Lipo@Cy3-siRNA/VP NPs obtained after liposome coating can effectively improve the stability of Cy3-siRNA/VP NPs, especially the improvement of its stability in serum, which is beneficial to its better function in the human body.
实施例6对前列腺肿瘤细胞生长的抑制效果考察。Example 6 Investigation of the inhibitory effect on the growth of prostate tumor cells.
1.不同浓度VP在光照后对前列腺肿瘤细胞生长的抑制效果考察1. Investigation of the inhibitory effect of different concentrations of VP on the growth of prostate tumor cells after light exposure
将DU145细胞接种于96孔板中，每组3个复孔，细胞孔最外围每孔加入100μL的PBS，放置于37℃、5％CO2培养箱培养；待细胞贴壁后，加入100μL含不同浓度VP(0，1，2，3，4μM)的培养基；避光培养15min后每孔用690nm激光光照120s；光照后继续孵育48h；吸弃96孔板中的培养基，加入PBS清洗2次，后加入100μL培养基与CCK8混合溶液(每孔10μL CCK8试剂+90μL培养基)，孵育1h后使用酶标仪测定培养液在450nm处的吸光度。通过计算得IC50值。DU145 cells were seeded in 96-well plates, with 3 replicate wells in each group, 100 μL of PBS was added to each well of the outermost cell wells, and placed in a 37°C, 5% CO2 incubator for culture; after the cells adhered to the wall, 100 μL of PBS containing different Concentration VP (0, 1, 2, 3, 4 μM) medium; after 15 minutes of dark culture, each well was illuminated with 690nm laser light for 120s; after illumination, the incubation was continued for 48 hours; the medium in the 96-well plate was discarded, and PBS was added to wash for 2 Then add 100 μL medium and CCK8 mixed solution (10 μL CCK8 reagent + 90 μL medium per well), and after incubation for 1 h, use a microplate reader to measure the absorbance of the culture solution at 450 nm. The IC50 value was obtained by calculation.
2.Lipo@Cy3-siRNA/VP NPs对前列腺肿瘤细胞生长的抑制效果考察，并与Cy3-siRNA、VP、Cy3-siRNA/VP NPs对比。2. The inhibitory effect of Lipo@Cy3-siRNA/VP NPs on the growth of prostate tumor cells was investigated, and compared with Cy3-siRNA, VP, and Cy3-siRNA/VP NPs.
根据上述实验所得VP的IC50值＝1.387μM。按Cy3-siRNA/VP＝1/30设置各组药物(Lipo@Cy3-siRNA/VP NPs、Cy3-siRNA、VP、Cy3-siRNA/VP NPs)中VP浓度为1.387μM，Cy3-siRNA浓度为：0.046μM。将DU145细胞接种于96孔板中，每组3个复孔，放置于37℃、5％CO2培养箱培养；待细胞贴壁后，分别加入Lipo@Cy3-siRNA/VP NPs(0.046μM/1.387μM)、Cy3-siRNA(0.046μM)、VP(1.387μM)、Cy3-siRNA/VP NPs(0.046μM/1.387μM)；避光培养15min后每孔用690nm激光光照120s；光照后继续孵育48h；吸弃96孔板中的培养基，加入PBS清洗2次，后加入100μL培养基与CCK8混合溶液(每孔10μL CCK8试剂+90μL培养基)，孵育1h后使用酶标仪测定培养液在450nm处的吸光度。According to the above experiment, the IC50 value of VP was 1.387 μM. According to Cy3-siRNA/VP=1/30, the concentration of VP in each group of drugs (Lipo@Cy3-siRNA/VP NPs, Cy3-siRNA, VP, Cy3-siRNA/VP NPs) is set to 1.387 μM, and the concentration of Cy3-siRNA is: 0.046 μM. DU145 cells were seeded in 96-well plates, with 3 replicate wells in each group, and placed in a 37°C, 5% CO2 incubator for culture; after the cells adhered to the wall, Lipo@Cy3-siRNA/VP NPs (0.046 μM/1.387 μM), Cy3-siRNA (0.046μM), VP (1.387μM), Cy3-siRNA/VP NPs (0.046μM/1.387μM); after 15 minutes of dark incubation, each well was illuminated with a 690nm laser for 120s; after illumination, the incubation was continued for 48h; Aspirate and discard the culture medium in the 96-well plate, add PBS to wash twice, then add 100 μL medium and CCK8 mixed solution (10 μL CCK8 reagent + 90 μL medium per well), incubate for 1 hour and use a microplate reader to measure the temperature of the culture medium at 450 nm of absorbance.
根据计算，得到细胞存活率及抑制率。计算公式如下：细胞存活率＝[(As-Ab)/(Ac-Ab)]*100％.抑制率＝100％-存活率。(As：实验孔吸光度(含细胞、培养液、CCK-8溶液和药物溶液)；Ac：对照孔吸光度(含细胞、培养基、CCK-8溶液、不含药物)；Ab：空白孔吸光度(含培养基、CCK-8溶液、不含细胞、药物)。)。According to the calculation, the cell viability and inhibition rate were obtained. The calculation formula is as follows: cell survival rate=[(As-Ab)/(Ac-Ab)]*100%. Inhibition rate=100%-survival rate. (As: absorbance of experimental well (containing cells, culture medium, CCK-8 solution and drug solution); Ac: absorbance of control well (containing cells, medium, CCK-8 solution, no drug); Ab: absorbance of blank well ( Contains culture medium, CCK-8 solution, does not contain cells, drugs).).
实验结果：我们首先探究不同浓度VP光照120s对于DU145细胞的增殖抑制能力，结果如图6中A所示，可以得出VP光照后对于DU145细胞的半数抑制浓度(IC50)为1.387μM。接下来，我们设置各组药物中的VP浓度为1.387μM，探究Lipo@Cy3-siSkp2/VP NPs抑制DU145细胞增殖的能力。我们分别检测了游离Cy3-siSkp2、游离VP、Cy3-siSkp2/VP NPs以及Lipo@Cy3-siSkp2/VP NPs治疗组DU145细胞的细胞活性。结果如图6中B所示，在VP浓度为1.387μM时，游离Cy3-siSkp2对于DU145细胞基本无抑制作用，Cy3-siRNA/VP NPs与游离VP对DU145细胞的抑制效果相似(分别为40.5％与51.7％)，可能时由于Cy3-siRNA/VP NPs在FBS中极不稳定、易解离，故其对DU145细胞的抑制作用效果与游离的VP组大致相同。而Lipo@Cy3-siSkp2/VP NPs组对于DU145细胞的活性抑制作用相比于其他组具有明显增强，抑制率高达90％，这得益于Lipo@Cy3-siSkp2/VP NPs粒子中的脂质体(Lipo)对于Cy3-siSkp2/VP NPs的保护作用，使其能够顺利进入DU145细胞，并发挥光动力治疗与基因联合治疗的作用效果，表现出对DU145细胞更好的抑制效果。Experimental results: We first explored the inhibitory ability of different concentrations of VP to illuminate the proliferation of DU145 cells for 120s. The results are shown in Figure 6A. It can be concluded that the half inhibitory concentration (IC50) of VP to DU145 cells after irradiation is 1.387 μM. Next, we set the concentration of VP in each group of drugs to be 1.387 μM to explore the ability of Lipo@Cy3-siSkp2/VP NPs to inhibit the proliferation of DU145 cells. We detected the cell viability of DU145 cells in the treatment groups of free Cy3-siSkp2, free VP, Cy3-siSkp2/VP NPs and Lipo@Cy3-siSkp2/VP NPs, respectively. The results are shown in Figure 6 B, when the concentration of VP was 1.387 μM, free Cy3-siSkp2 had no inhibitory effect on DU145 cells, and the inhibitory effects of Cy3-siRNA/VP NPs and free VP on DU145 cells were similar (40.5% respectively and 51.7%), possibly because Cy3-siRNA/VP NPs is extremely unstable and easily dissociated in FBS, so its inhibitory effect on DU145 cells is roughly the same as that of the free VP group. Compared with other groups, the inhibitory effect of Lipo@Cy3-siSkp2/VP NPs group on the activity of DU145 cells was significantly enhanced, and the inhibition rate was as high as 90%, which was due to the liposomes in Lipo@Cy3-siSkp2/VP NPs particles (Lipo) has a protective effect on Cy3-siSkp2/VP NPs, enabling it to enter DU145 cells smoothly, and play the role of photodynamic therapy and gene therapy, showing a better inhibitory effect on DU145 cells.
虽然以上描述了本发明的具体实施方式，但是熟悉本技术领域的技术人员应当理解，我们所描述的具体的实施例只是说明性的，而不是用于对本发明的范围的限定，熟悉本领域的技术人员在依照本发明的精神所作的等效的修饰以及变化，都应当涵盖在本发明的权利要求所保护的范围内。Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments we have described are only illustrative, rather than used to limit the scope of the present invention. Equivalent modifications and changes made by skilled personnel in accordance with the spirit of the present invention shall fall within the protection scope of the claims of the present invention.
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