靶向纳米生物复合物在癌细胞成像诊断分析、药物输送以及治疗中的研究与应用
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摘要
恶性肿瘤(癌症),是当今严重威胁人类健康的主要疾病之一,早期发现并及时治疗是治疗肿瘤的关键。目前的常规诊断方法灵敏度和特异性比较低,在肿瘤发生早期很难检测,大多数患者在被确诊时肿瘤细胞已发生浸润和转移,错过了治疗的有效时机。尽管近年来随着药物和治疗方法的不断革新,临床的治疗效果已经取得了很大的进步,但是目前有效的治疗方法依然是手术、放疗和化疗等传统方法。尤其是化疗,大量的研究集中在提高化疗的疗效上,因为化疗被认为是最有希望治愈癌症的方法。然而目前化疗遭遇了很多挑战,例如靶向输送效果差、强烈的毒副作用以及肿瘤细胞的耐药性等,难以达到预期的治疗效果。因此,发展灵敏度高、特异性强的早期诊断方法以及开发具有高的肿瘤靶向性、安全、有效、经济的抗肿瘤药物体系已经成为目前癌症研究的热点,对癌症的早期诊断和治疗有着重要的意义。
基于纳米材料的荧光探针及药物载体已经被广泛地应用于生物化学领域,特别是肿瘤的成像诊断及治疗。这其中,金纳米粒子因具有良好的生物相容性使得其在生物标记、成像以及药物输送等方面引起了人们的广泛关注,基于纳米金的探针以及药物载体也已经被大量报道。然而目前的大多数探针只能检测一种肿瘤特异标记物,在肿瘤的诊断中常常会造成“假阳性”结果。而且药物载体不能根据肿瘤的实时发展状况可控地释放药物,为肿瘤患者提供合理的药物剂量,从而引起过多用药,造成了严重的毒副作用。此外,传统的单一疗法还不能取得令人满意的治疗效果,因此发展灵敏的、特异的新型纳米荧光探针对肿瘤细胞内的多种特异标记物诊断以及发展理性可控的药物载体靶向输送药物到肿瘤,并开展联合疗法来杀死肿瘤细胞已成为目前抗癌领域的研究热点。
本论文基于生物相容性非常好的纳米金、分子信标、广谱抗癌药物阿霉素以及金纳米棒和光敏剂组装了一系列纳米荧光探针和纳米药物载体用于癌细胞的成像诊断分析,药物靶向输送、可控释放以及联合治疗等,主要包括:
1、基于纳米金(AuNP)和分子信标(MB),将靶向识别乳腺癌细胞中特异肿瘤标记物(细胞周期蛋白D1 mRNA,cyclin D1 mRNA)的分子信标组装到纳米金上得到AuNP-MB,并将Dox物理地装载到AuNP-MB上,发展了一种新奇的纳米药物载体AuNP-MB(Dox)。AuNP-MB(Dox)对cyclin D1 mRNA高表达的癌细胞具有高选择性和特异性,可对癌细胞进行荧光成像诊断分析和药物靶向输送,能有效地杀死癌细胞,而对正常细胞无伤害。重要的是,AuNP-MB(Dox)可根据癌细胞中cyclin D1 mRNA的量可控地释放不同量的Dox,即Dox的释放量和cyclin D1 mRNA的表达水平是正相关的。这种肿瘤特异mRNA依赖的药物释放载体可根据癌细胞的实时发展状况来释放合理剂量的Dox,从而减少了过多用药造成的毒副作用。
2、基于纳米金(AuNP)和双分子信标bi-MB(MB1,MB2),将靶向于乳腺癌细胞中两种特异肿瘤标记物的双bi-MB组装到AuNP上,发展了一种新奇的纳米双荧光探针AuNP-bi-MB。AuNP-bi-MB可对癌细胞中高表达的两种特异肿瘤mRNA(survivin mRNA,cyclin D1 mRNA)同时进行双色荧光成像及诊断分析,有效地避免了单一肿瘤mRNA检测技术中出现的“假阳性”结果,有利于肿瘤的早期诊断。重要的是可通过实时检测两种肿瘤特异mRNA的相对表达水平,来更加为明确肿瘤的发展状况,为肿瘤的有效治疗提供了可靠的信息。
3、基于金纳米棒(GNR)、分子信标(MB)和光敏剂,MB可靶向识别乳腺癌细胞中的特异肿瘤标记物生存素(survivin)mRNA,首先在MB的一端上修饰了Ce6得到MB-Ce6,然后将MB-Ce6组装到金纳米棒上,得到多功能的光控纳米药物载体GNR-MB-Ce6。GNR-MB-Ce6可靶向识别癌细胞内的特异标记物survivin mRNA并对癌细胞进行荧光成像及诊断分析,然后在近红外光的照射下,Ce6释放出单线态氧杀死癌细胞,同时金纳米棒的光热效应也可以杀死癌细胞,从而对癌细胞进行联合治疗,避免了单一疗法的局限性。
Malignant tumor (cancer) has a reputation as a deadly disease, and successful cure of most cancers depends on early detection and timely treatment. It is difficult to discover tumor in the early stage, because of the relatively low sensitivity and specificity of conventional diagnostic methods. Usually, the undetected metastasis has already occurred in patients before initial diagnosis, which leads to missing the opportunity to cure disease. Although the clinical arsenal in treating cancer has been greatly extended in recent years with the application of new drugs and therapeutic modalities, the three main approaches still to be surgical resection, radiation, and chemotherapy. Most research focus on how to improve the efficacy of chemotherapy which is a promising approach in cancer therapy. The challenges encountered by chemotherapy make it still difficult to achieve the desired therapeutic effect, including untargeted transport, serious side effects on healthy tissue and drug resistance. Therefore, it is significant to develop simple, sensitive and effective detection approaches and targeted, safe, effective antitumor drugs system for cancer diagnosis and therapy.
Fluorescent probes and drug carriers based on nano-materials has been widely used in biochemistry, especially in imaging diagnosis and treatment of cancer. The gold nanoparticle (AuNP) has an extraordinary intracellular stability and biocompatibility that makes it useful for intracellular imaging, molecular diagnostics and drug delivery. AuNP-based probes and drug carriers has been widely reported. However, most current probes detect usually a single tumor marker, which often result in "false positive" results for cancer diagnosis. The realease of drug from delivery carrier lacks the consistent correlations with tumor progression and usually results in unpredictable release, resulting in serious side effects. In addition, the single traditional therapy can not achieve satisfactory therapeutic effects. Thus, sensitive and specific fluorescent probes for simultaneous detecting several tumor markers, controlled and targeted drug carrier and combined antitumor therapy have become research focus in the field of cancer diagnosis and therapy.
In this study, based on the biocompatible AuNP, molecular beacon (MB), antitumor Doxorubicin (Dox), the gold nanorod (GNR) and photosensitizer (PS), we developed a series of fluorescent nanoprobes and drug nano-carriers for cancer diagnosis and therapy as follows:
1. We demonstrated an effective and controlled drug carrier for the delivery of Dox based on AuNP-MB. It could target cyclin D1 mRNA, which is a tumor mRNA of breast cancer. When MB bound selectively to cyclin D1 mRNA, fluorescent Dox released effectively from AuNP–MB(Dox) and induced apoptosis, which occurred in breast cancer cells (cyclin D1+) but not in normal cells (cyclin D1-). Significantly, the release of Dox was correlated positively with the quantities of tumor mRNA. This drug carrier released Dox according to various stages of tumor progression in a controlled manner which could decrease effectively side effects of Dox.
2. Based on AuNP and bi-MB, we developed a bi-color fluorescence imaging agent AuNP-bi-MB to detect simultaneously intracellular tumor mRNAs in breast cancer. The AuNP was assembled with bi-MB which targeted specifically to two kinds of tumor mRNAs. This novel approach could prevent effectively“false positive”results and provide comprehensive and dependable information for early detection of cancer compared with single tumor mRNA testing. It would be beneficial to identify the stage of tumor progression and assess treatment decisions by real-time detection of relative expression level of tumor mRNAs in cancer cells.
3. Based on GNR, MB and PS, we developed a light-triggered drug nano-carrier GNR-MB-Ce6 targeting survivin mRNA which is a tumor mRNA of breast cancer. GNR-MB-Ce6 was assembled by Ce6-labeled MB and GNR. GNR-MB-Ce6 could detect survivin mRNA and be used in cancer cell imaging. Under the irradiation of NIR light, Ce6 generated singlet oxygen as photodynamic theray (PDT) agent, GNR generated photothermal effect and damage cancer cell. Combination of PDT and photothermal effect could induce effectively apoptosis in cancer cell and avoid the limitations of single therapy.
基于纳米材料的荧光探针及药物载体已经被广泛地应用于生物化学领域,特别是肿瘤的成像诊断及治疗。这其中,金纳米粒子因具有良好的生物相容性使得其在生物标记、成像以及药物输送等方面引起了人们的广泛关注,基于纳米金的探针以及药物载体也已经被大量报道。然而目前的大多数探针只能检测一种肿瘤特异标记物,在肿瘤的诊断中常常会造成“假阳性”结果。而且药物载体不能根据肿瘤的实时发展状况可控地释放药物,为肿瘤患者提供合理的药物剂量,从而引起过多用药,造成了严重的毒副作用。此外,传统的单一疗法还不能取得令人满意的治疗效果,因此发展灵敏的、特异的新型纳米荧光探针对肿瘤细胞内的多种特异标记物诊断以及发展理性可控的药物载体靶向输送药物到肿瘤,并开展联合疗法来杀死肿瘤细胞已成为目前抗癌领域的研究热点。
本论文基于生物相容性非常好的纳米金、分子信标、广谱抗癌药物阿霉素以及金纳米棒和光敏剂组装了一系列纳米荧光探针和纳米药物载体用于癌细胞的成像诊断分析,药物靶向输送、可控释放以及联合治疗等,主要包括:
1、基于纳米金(AuNP)和分子信标(MB),将靶向识别乳腺癌细胞中特异肿瘤标记物(细胞周期蛋白D1 mRNA,cyclin D1 mRNA)的分子信标组装到纳米金上得到AuNP-MB,并将Dox物理地装载到AuNP-MB上,发展了一种新奇的纳米药物载体AuNP-MB(Dox)。AuNP-MB(Dox)对cyclin D1 mRNA高表达的癌细胞具有高选择性和特异性,可对癌细胞进行荧光成像诊断分析和药物靶向输送,能有效地杀死癌细胞,而对正常细胞无伤害。重要的是,AuNP-MB(Dox)可根据癌细胞中cyclin D1 mRNA的量可控地释放不同量的Dox,即Dox的释放量和cyclin D1 mRNA的表达水平是正相关的。这种肿瘤特异mRNA依赖的药物释放载体可根据癌细胞的实时发展状况来释放合理剂量的Dox,从而减少了过多用药造成的毒副作用。
2、基于纳米金(AuNP)和双分子信标bi-MB(MB1,MB2),将靶向于乳腺癌细胞中两种特异肿瘤标记物的双bi-MB组装到AuNP上,发展了一种新奇的纳米双荧光探针AuNP-bi-MB。AuNP-bi-MB可对癌细胞中高表达的两种特异肿瘤mRNA(survivin mRNA,cyclin D1 mRNA)同时进行双色荧光成像及诊断分析,有效地避免了单一肿瘤mRNA检测技术中出现的“假阳性”结果,有利于肿瘤的早期诊断。重要的是可通过实时检测两种肿瘤特异mRNA的相对表达水平,来更加为明确肿瘤的发展状况,为肿瘤的有效治疗提供了可靠的信息。
3、基于金纳米棒(GNR)、分子信标(MB)和光敏剂,MB可靶向识别乳腺癌细胞中的特异肿瘤标记物生存素(survivin)mRNA,首先在MB的一端上修饰了Ce6得到MB-Ce6,然后将MB-Ce6组装到金纳米棒上,得到多功能的光控纳米药物载体GNR-MB-Ce6。GNR-MB-Ce6可靶向识别癌细胞内的特异标记物survivin mRNA并对癌细胞进行荧光成像及诊断分析,然后在近红外光的照射下,Ce6释放出单线态氧杀死癌细胞,同时金纳米棒的光热效应也可以杀死癌细胞,从而对癌细胞进行联合治疗,避免了单一疗法的局限性。
Malignant tumor (cancer) has a reputation as a deadly disease, and successful cure of most cancers depends on early detection and timely treatment. It is difficult to discover tumor in the early stage, because of the relatively low sensitivity and specificity of conventional diagnostic methods. Usually, the undetected metastasis has already occurred in patients before initial diagnosis, which leads to missing the opportunity to cure disease. Although the clinical arsenal in treating cancer has been greatly extended in recent years with the application of new drugs and therapeutic modalities, the three main approaches still to be surgical resection, radiation, and chemotherapy. Most research focus on how to improve the efficacy of chemotherapy which is a promising approach in cancer therapy. The challenges encountered by chemotherapy make it still difficult to achieve the desired therapeutic effect, including untargeted transport, serious side effects on healthy tissue and drug resistance. Therefore, it is significant to develop simple, sensitive and effective detection approaches and targeted, safe, effective antitumor drugs system for cancer diagnosis and therapy.
Fluorescent probes and drug carriers based on nano-materials has been widely used in biochemistry, especially in imaging diagnosis and treatment of cancer. The gold nanoparticle (AuNP) has an extraordinary intracellular stability and biocompatibility that makes it useful for intracellular imaging, molecular diagnostics and drug delivery. AuNP-based probes and drug carriers has been widely reported. However, most current probes detect usually a single tumor marker, which often result in "false positive" results for cancer diagnosis. The realease of drug from delivery carrier lacks the consistent correlations with tumor progression and usually results in unpredictable release, resulting in serious side effects. In addition, the single traditional therapy can not achieve satisfactory therapeutic effects. Thus, sensitive and specific fluorescent probes for simultaneous detecting several tumor markers, controlled and targeted drug carrier and combined antitumor therapy have become research focus in the field of cancer diagnosis and therapy.
In this study, based on the biocompatible AuNP, molecular beacon (MB), antitumor Doxorubicin (Dox), the gold nanorod (GNR) and photosensitizer (PS), we developed a series of fluorescent nanoprobes and drug nano-carriers for cancer diagnosis and therapy as follows:
1. We demonstrated an effective and controlled drug carrier for the delivery of Dox based on AuNP-MB. It could target cyclin D1 mRNA, which is a tumor mRNA of breast cancer. When MB bound selectively to cyclin D1 mRNA, fluorescent Dox released effectively from AuNP–MB(Dox) and induced apoptosis, which occurred in breast cancer cells (cyclin D1+) but not in normal cells (cyclin D1-). Significantly, the release of Dox was correlated positively with the quantities of tumor mRNA. This drug carrier released Dox according to various stages of tumor progression in a controlled manner which could decrease effectively side effects of Dox.
2. Based on AuNP and bi-MB, we developed a bi-color fluorescence imaging agent AuNP-bi-MB to detect simultaneously intracellular tumor mRNAs in breast cancer. The AuNP was assembled with bi-MB which targeted specifically to two kinds of tumor mRNAs. This novel approach could prevent effectively“false positive”results and provide comprehensive and dependable information for early detection of cancer compared with single tumor mRNA testing. It would be beneficial to identify the stage of tumor progression and assess treatment decisions by real-time detection of relative expression level of tumor mRNAs in cancer cells.
3. Based on GNR, MB and PS, we developed a light-triggered drug nano-carrier GNR-MB-Ce6 targeting survivin mRNA which is a tumor mRNA of breast cancer. GNR-MB-Ce6 was assembled by Ce6-labeled MB and GNR. GNR-MB-Ce6 could detect survivin mRNA and be used in cancer cell imaging. Under the irradiation of NIR light, Ce6 generated singlet oxygen as photodynamic theray (PDT) agent, GNR generated photothermal effect and damage cancer cell. Combination of PDT and photothermal effect could induce effectively apoptosis in cancer cell and avoid the limitations of single therapy.
引文
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