Review 评论

Functional nanoparticles for molecular imaging guided gene delivery
分子影像引导基因传递的功能性纳米颗粒

Introduction 介绍

Gene therapy has shown potential to treat human diseases that occur from defective genes like cystic fibrosis, macular degeneration, Parkinson's disease, and different types of cancers [1], [2], [3], [4]. The development of efficient gene therapy depends on an efficient transfer of therapeutic genes into a cell to replace or silence defective ones associated with human disease. Viral vectors like adenoviruses and retroviruses are commonly used in gene therapy due to their high efficiency of gene delivery. However, there are several recurring issues that have led to a reconsideration of the use of viral vectors in human clinical trials, such as immunological problems, insertional mutagenesis and limitations in the size of the carried therapeutic genes.
基因治疗已显示出潜力，可以治疗由缺陷基因引起的人类疾病，如囊性纤维化、黄斑变性、帕金森病和不同类型的癌症。有效的基因治疗的发展取决于将治疗基因有效地传递到细胞中，以取代或沉默与人类疾病相关的有缺陷的基因。腺病毒和逆转录病毒等病毒载体通常用于基因治疗，因为它们具有高效的基因传递能力。然而，在人类临床试验中使用病毒载体出现了一些反复出现的问题，因此人们开始重新考虑病毒载体的使用，比如免疫问题、插入突变原性和携带治疗基因大小的限制。

Recently, non-viral particles have been receiving increasing attention in gene therapy, since they can overcome major viral delivery toxicity issues [5]. Common non-viral vectors that allow the genetic material to pass through cellular barriers are extensively discussed elsewhere [6], [7], [8], [9]. However, it remains a great challenge to find a carrier that will (1) load genetic materials, (2) pass the material through cellular barriers without causing a foreign body immune response, (3) release it into the cell nucleus, and (4) allow the visualization of this entire process without degrading the genetic materials. Other factors affect the effectiveness of gene therapy like the short-lived nature of the therapeutic DNA within the dividing cells and the multigene nature of many disorders where numerous mutations occur on many genes. In addition to such issues, the effectiveness of gene therapy is difficult to study without visualizing the exact transport noninvasively. Therefore there is an urgent need to develop sensitive and noninvasive methods that could be performed to overcome the challenges of gene therapy such as utilizing nano-dimensional materials to carry genes across cellular membrane barriers and exploiting unique optical or magnetic properties for noninvasive and spatiotemporal molecular imaging of gene delivery.
近年来，非病毒颗粒在基因治疗中受到越来越多的关注，因为它们能够克服主要的病毒传递毒性问题[5]。允许遗传物质通过细胞屏障的常见非病毒载体在其他地方已经广泛讨论[6]、[7]、[8]、[9]。然而，找到一种载体仍然是一个巨大的挑战，这种载体能够（1）装载遗传物质，（2）在不引起外源性免疫反应的情况下通过细胞屏障，（3）将其释放到细胞核中，以及（4）允许对整个过程进行可视化而不使遗传物质降解。其他因素影响基因治疗的有效性，比如治疗性 DNA 在分裂细胞内的短寿命以及许多多基因失调疾病的多基因性质，其中许多基因上发生许多突变。除了这些问题，基因治疗的有效性很难在不可逆无创的情况下进行研究。因此，迫切需要开发敏感和无创的方法，以克服基因治疗的挑战，如利用纳米材料跨越细胞膜屏障携带基因，并利用独特的光学或磁性能进行无创和时空分子成像的基因传递。

Molecular imaging has flourished over the last decade. Advanced molecular imaging techniques for gene therapy monitoring will enable real-time assessment of the therapeutic process and the refinement of current gene therapy protocols. Probes can allow either direct or indirect spatiotemporal evaluation of gene delivery and gene expression utilizing molecular imaging methods to guide therapeutic gene delivery and monitor the therapeutic response [10], [11]. Through non-invasive monitoring of the distribution and kinetics of vector-mediated gene expression, molecular imaging can provide the functionality and most importantly the efficacy of vector and gene delivery systems. Molecular imaging is likely to aid in an improved design of targeted gene transfer methods and the selection and development of safe and efficient gene delivery systems.
分子影像学在过去的十年中蓬勃发展。用于基因治疗监测的先进分子影像技术将实现对治疗过程的实时评估，并改进当前基因治疗方案。探针能够直接或间接进行基因传递和基因表达的时空评估，利用分子影像方法指导治疗基因传递并监测治疗反应。通过非侵入性监测载体介导的基因表达的分布和动力学，分子影像能够提供载体和基因传递系统的功能性和更重要的是效力。分子影像很可能有助于改进靶向基因转移方法的设计，以及选择和开发安全高效的基因传递系统。

The emergence of molecular imaging strategies has been pivotal in optimizing gene therapy with advanced probes [12], [13]. Currently, one typical probe for molecular imaging in gene therapy is a unified fusion gene composed of both the therapy and imaging reporter gene whose expression can be imaged using multiple modalities [14]. This strategy is very useful to determine the patterns of gene expression that encode the biological processes of diseases. To date, there have been many imaging reporter genes used in the field of reporter gene imaging, such as herpes simplex virus type 1 thymidine kinase gene for single photon emission computed tomography (SPECT) and positron emission tomography (PET) [14], [15], transferrin receptor gene for magnetic resonance imaging (MRI) [16], and fluorescent protein gene for optical imaging [12], [15]. Generally, imaging reporter genes are used to study promoter or enhancer elements involved in disease-related gene expression. A promoter of a specific disease biomarker is inserted and the molecular imaging reporter gene is placed under the control of the special promoter fragments. The promoter can be inducible/constitutive and cell-specific and transcription of the reporter gene can be tracked, allowing the study of gene expression. The ideal imaging reporter genes would have the following characteristics: lack of immune response, favorable kinetics, stability and biocompatibility. However, no reporter gene has been found that meets all these criteria at present.
分子成像策略的出现对优化基因治疗至关重要，配备高级探针[12]，[13]。目前，基因治疗中一种典型的分子成像探针是由治疗和成像报告基因组成的统一融合基因，其表达可以用多种成像模式进行观察[14]。这种策略非常有用，可以确定编码疾病生物过程的基因表达模式。迄今为止，在分子成像报告基因成像领域已经使用了许多成像报告基因，例如单光子发射计算机断层摄影（SPECT）和正电子发射计算机断层摄影（PET）的单纯疱疹病毒 1 型胸腺苷激酶基因[14]，[15]，转铁蛋白受体基因用于磁共振成像（MRI）[16]，以及荧光蛋白基因用于光学成像[12]，[15]。通常使用成像报告基因来研究与疾病相关基因表达有关的启动子或增强子元素。将特定疾病生物标志物的启动子插入，并将分子成像报告基因置于特殊启动子片段的控制下。启动子可以是可诱导的/构成性的和细胞特异性的，可以跟踪报告基因的转录，以便研究基因表达。理想的成像报告基因应具备以下特点：缺乏免疫反应，动力学良好，稳定性和生物兼容性好。然而，到目前为止还没有发现符合所有这些标准的报告基因。

Another strategy to make molecular imaging probes and gene delivery vehicles is based on nanomaterials [17], [18]. Emerging nanomaterials provide platforms that have various sizes and structures that may be used to develop nanoparticles (NPs) with the capability to serve as gene delivery vectors and molecular imaging agents (Fig. 1). At present, there are several types of NPs available for gene therapy and molecular imaging. Such NP-based imaging probes afford many advantages over conventional small-molecule-based approaches [17], [19], [20], [21], [22]. For example, the ease of functionalizing the NP surface is a clear advantage in designing molecular carrier and probes. Imaging labels (fluorescence tags, radionuclides, and other biomolecules) or a combination of labels for different imaging modalities can be attached to a single NP, which can lead to dramatic signal amplification. Furthermore, targeting motifs, such as antibodies, peptides, aptamers, and small molecules, on the nanoparticle can provide enhanced binding affinity and specificity. The roles of molecular imaging in gene therapy continue to increase because of advances in imaging technologies and concomitant improvements in detection sensitivity and specificity with functional NPs. The combination of different targeting ligands, imaging labels, genetically engineered genes, and many other agents may allow for effective and controlled gene delivery, that could be noninvasively and quantitatively monitored in real time. These multifunctional systems will enhance diagnostic evaluation and gene therapy development and predict clinical outcomes, fulfilling the promise of personalized and advanced medicine. In the subsequent sections we discuss the functional NPs and systems for molecular imaging guided gene delivery and highlight some of the most advanced examples.
基于纳米材料的分子成像探针和基因传递载体的另一种策略是新兴纳米材料提供了各种尺寸和结构的平台，可用于开发能够作为基因传递载体和分子成像剂的纳米颗粒（NPs）。目前，有几种类型的 NPs 可用于基因治疗和分子成像。这些基于 NP 的成像探针比传统的小分子方法具有许多优势。例如，易于功能化 NP 表面的优点在设计分子载体和探针时非常明显。成像标签（荧光标记、放射性核素和其他生物分子）或不同成像方式的标记组合可以附着在单个 NP 上，这可以导致信号的显著增强。此外，纳米颗粒上的靶向基序（如抗体、肽、适配体和小分子）可以提供增强的结合亲和力和特异性。分子成像在基因治疗中的作用由于成像技术的进展以及功能性 NPs 对检测灵敏度和特异性的改善而不断增强。不同靶向配体、成像标签、基因工程基因和许多其他试剂的组合可以实现有效和可控的基因递送，并可实时进行无创和定量监测。这些多功能系统将增强诊断评估和基因治疗的发展，并预测临床结果，实现个性化和先进医学的承诺。 在接下来的部分中，我们讨论了用于分子成像引导基因传递的功能性 NP 和系统，并突出了一些最先进的示例。

1. Download : Download high-res image (412KB)
   下载：下载高清晰度图片（412KB）
2. Download : Download full-size image
   下载：下载全尺寸图像

Figure 1. Schematic diagram of functional nanoparticles for molecular imaging guided gene delivery.
图 1. 功能性纳米粒子的示意图，用于分子影像引导基因传递。

Polymer-based nanoparticles
基于聚合物的纳米颗粒

Cationic polymers can form stable polyplexed NPs with DNAs through electrostatic interactions and these polycation/DNA complexes are by far the most widely used non-viral gene delivery vectors. Many factors affect gene transfection efficiency of polymer-based NPs including molecular weight, surface charges, amphiphilicity and the structure and shape of NPs. Several cationic polymers such as polyethyleneimine (PEI), poly-l-lysine (PLL), chitosan and poly(amidoamines) (PAMAM) are used as important vectors for gene delivery. To date, various nanobioconjugation techniques have been reported to enhance gene transfection efficiency and to reduce toxicity of theses polymers in vitro and in vivo. Several comprehensive review articles have summarized the applications of cationic polymer-based gene therapy [23], [24], [25]. Imaging techniques allow the visualization of important issues involving polymeric nanoparticles for gene delivery. For example, optical imaging could visualize the degradation of polymers [26], the cellular uptake and secretion of genes and polymers [27], [28], and the dissociation of genes and polymers after uptake [28], [29]. In this section, a few recent examples of polymer-based gene carriers accompanied by molecular imaging techniques will be introduced.
阳离子聚合物可以通过静电相互作用与 DNA 形成稳定的聚阳离子复合纳米粒子，这些聚阳离子/DNA 复合物迄今为止是最广泛使用的非病毒基因传递载体。许多因素影响基于聚合物的纳米粒子的基因转染效率，包括分子量、表面电荷、亲疏水性以及纳米粒子的结构和形状。多种阳离子聚合物，如聚乙烯亚胺（PEI）、聚-L-赖氨酸（PLL）、壳聚糖和聚（酰胺胺）（PAMAM）被用作重要的基因传递载体。迄今为止，已有多种纳米生物共轭技术报告，以增强基因转染效率并在体外和体内减少这些聚合物的毒性。一些综合性评论文章总结了基于阳离子聚合物的基因疗法的应用。成像技术允许可视化涉及用于基因传递的聚合物纳米粒子的重要问题。例如，光学成像可以观察到聚合物的降解，基因和聚合物的细胞摄取和分泌，以及摄取后基因和聚合物的解离。在本节中，将介绍几个最近的基于聚合物的基因载体例子，辅以分子影像技术。

Yao et al. reported the use of folate grafted PEI₆₀₀-cyclodextrins (folate-PEI₆₀₀-CyD) as an effective polyplex-forming plasmid delivery agent with low toxicity [30]. The toxicity of PEI was reduced by the grafting of β-cyclodextrin as previously reported [31]. To further improve the efficiency of PEI₆₀₀-CyD delivery, folic acid was conjugated as the targeting ligand. Folate-PEI₆₀₀-CyD was five-fold less toxic compared to that of PEI-25 kDa in U87 cells and showed the highest transfection efficiency, about 60–70%, in folic receptor over-expressing U138 and U87 cells. In vivo bioluminescence imaging, made possible by the luciferase plasmid (pLuc), showed that the efficiency of folate-PEI₆₀₀-CyD-mediated transfection was 13-fold higher compared to pLuc alone and was comparable to that of adenovirus-mediated luciferase transduction in melanoma-bearing mice.
Yao 等报道了叶酸接枝的 PEI ₆₀₀ -环糊精（叶酸-PEI ₆₀₀ -CyD）作为一种低毒性的有效聚合物复合体形成质粒传输剂[30]。PEI 的毒性通过接枝β-环糊精得以减少，这已有文献报道[31]。为了进一步提高 PEI ₆₀₀ -CyD 递送的效率，将叶酸做为靶向配体进行了共轭。叶酸-PEI ₆₀₀ -CyD 在 U87 细胞中的毒性是 PEI-25kDa 的五分之一，并在表达叶酸受体的 U138 和 U87 细胞中展现了最高的转染效率，达到 60-70%左右。通过体内生物发光成像技术，利用荧光酶质粒（pLuc），叶酸-PEI ₆₀₀ -CyD 介导的转染效率比独立 pLuc 高出 13 倍，与腺病毒介导的荧光酶转导在携带黑色素的小鼠中类似。

A polyelectrolyte complex (PEC) micelle-based siRNA delivery system was developed for anti-angiogenic gene therapy and imaging [32]. The charge complexation between PEG-conjugated vascular endothelial growth factor siRNA (VEGF siRNA-PEG) and PEI led to the spontaneous formation of nanoscale PEC micelles, having a characteristic siRNA/PEI PEC inner core with a surrounding PEG shell layer. Intravenous and intratumoral injection of the siRNA containing PEC micelles significantly inhibited VEGF expression at the tumor tissue and suppressed tumor growth in an animal model without showing detectable inflammatory responses in mice. To further confirm the accumulation of PEC micelles in the solid tumor region, the siRNA was labeled with the near-infrared dye Cy5.5 and optically imaged in vivo. The siRNA-PEG/PEC micelles predominantly accumulated in the tumor, affirming the feasibility of siRNA-based gene therapy and imaging.
已开发出基于聚电解质复合物（PEC）胶束的 siRNA 递送系统，用于抗血管生成基因治疗和成像。PEG 共轭的血管内皮生长因子 siRNA（VEGF siRNA-PEG）与 PEI 之间的电荷结合导致纳米级 PEC 胶束的自发形成，具有具有特征的 siRNA/PEI PEC 内核和周围 PEG 外壳层。在动物模型中，静脉注射和肿瘤内注射含 siRNA 的 PEC 胶束显著抑制了肿瘤组织中 VEGF 表达，并抑制了肿瘤生长，而对小鼠没有检测到炎症反应。为进一步确认 PEC 胶束在固体肿瘤区域的积累，将 siRNA 标记为近红外染料 Cy5.5，并在体内进行光学成像。siRNA-PEG/PEC 胶束主要积聚在肿瘤中，证实了 siRNA 基因治疗和成像的可行性。

Another PEC for siRNA delivery was designed by low molecular weight chitosan (CS, m.w. 50–150 kDa) derivatized with poly-l-arginine (PLR) and PEG [33]. Among several cationic polymers, CS has been widely studied in delivery systems of negatively charged nucleic acid-based medicine due to its biocompatibility, biodegradability and low cost [34]. The PRL-grafted CS formed PEC, with a size less than 400 nm, showed higher cellular delivery efficiency of siRNA over unmodified CS, PEGylated CS, unmodified PLR, or PEGylated PLR. In vivo imaging of transfection efficiency after siRNA treatment was studied by using mice bearing B16F10-RFP (red fluorescent protein) tumors. Optical imaging revealed that the intratumoral administration of RFP-specific siRNA complexed with PEGylated CS-PLR significantly silenced the expression of RFPs in tumor tissue in vivo by 90% as compared to that in untreated tumor tissues.
一种用于 siRNA 传递的另一种低分子量壳聚糖（分子量 50-150kDa）被设计为与聚赖氨酸（PLR）和 PEG 进行衍生[33]。在几种阳离子聚合物中，由于其生物相容性、生物降解性和低成本，壳聚糖已广泛应用于负电荷核酸药物传递系统的研究[34]。PRL 接枝的壳聚糖形成 PEC，尺寸小于 400nm，显示出 siRNA 的细胞传递效率高于未修饰的壳聚糖、聚乙二醇化的壳聚糖、未修饰的 PLR 或聚乙二醇化的 PLR。使用携带 B16F10-RFP（红色荧光蛋白）肿瘤的小鼠进行治疗后的转染效率体内成像研究。光学成像显示，与未经处理的肿瘤组织相比，使用 PEG 化的 CS-PLR 复合物给荷 RFP 特异性 siRNA 进行肿瘤内给药，在体内显着沉默了肿瘤组织中 RFPs 的表达，沉默效率达 90%。

Multifunctional NPs for simultaneous gene delivery and imaging can also be prepared in a convenient way using amphiphilic biopolymers. Liu et al. constructed bifunctional NPs by encapsulating hydrophobic Re(phen) complexes with a novel amphiphilic block copolymer, PDMAEMA-b-poly(PEGMA)-b-PDMAEMA, for simultaneous cell imaging and gene transfection in living cells [35]. Certain transition metal complexes, such as Ru and Re complexes, may eliminate short-lived autofluorescence and photobleaching by taking advantage of large Stokes shifts, high photostability, low energy absorption and relatively long fluorescence lifetimes [35]. The stable, water soluble and non-toxic NPs with red emission had good interaction with DNA, making them good biolablels for cell imaging and gene transfection.
利用两亲生物高分子，可以方便地制备用于同时基因传递和成像的多功能纳米颗粒。Liu 等人利用一种新型两亲嵌段共聚物 PDMAEMA-b-聚(PEGMA)-b-PDMAEMA，封装疏水 Re(phen)配合物构建了双功能纳米颗粒，能在活细胞中实现细胞成像和基因转染[35]。某些过渡金属配合物，如 Ru 和 Re 配合物，可利用大的 Stokes 位移、高的光稳定性、低的能量吸收以及相对较长的荧光寿命来消除短寿命的自发荧光和光漂白[35]。这些稳定、水溶性和无毒的红色发射纳米颗粒与 DNA 具有良好的相互作用，使其成为细胞成像和基因转染的良好生物标志物。

Huh et al. designed a new nanosized siRNA carrier systems composed of amphiphilic glycol chitosan (GC) and strongly positively charged PEI (Fig. 2) [36]. In order to prepare stable and tumor-homing NPs, each polymer was modified with hydrophobic 5β-cholanic acid and mixed to form self-assembled GC-PEI NPs via strong hydrophobic interactions of hydrophobic 5β-cholanic acids in the polymer. The siRNA encapsulated NPs formed compact and stable NP structures (250 nm in diameter) and exhibited rapid time-dependent cellular uptake and gene silencing profiles in B16F10-RFP cells. To determine if the siRNA-GC-PEI NPs specifically target tumors in vivo, Cy5.5-labeled siRNA containing NPs were monitored after intravenous injection in mice bearing SCC7 tumors. Cy5.5-siRNA-GC-PEI NPs showed a strong NIR fluorescence signal in tumor tissue within 1 h of injection and the signal persisted for up to two days, indicating the high tumor-targeting ability of NPs. Furthermore, NPs provided a significant inhibition of RFP gene expression of B16F10-RFP tumor-bearing mice, because of the high tumor-targeting ability.
Huh 等人设计了一种新的纳米级 siRNA 载体系统，由两性亲水性的聚乙醇胺修饰的壳寡糖(GC)和强阳离子的 PEI 组成（图 2）。为了制备稳定且靶向肿瘤的纳米粒子，每种聚合物均经 5β-胆甾酸疏水修饰，并混合以形成通过聚合物中疏水 5β-胆甾酸之间的强烈疏水相互作用自组装的 GC-PEI 纳米粒子。封装 siRNA 的纳米粒子形成了紧凑稳定的纳米粒子结构（直径 250nm），在 B16F10-RFP 细胞中表现出快速时间依赖的细胞摄取和基因沉默特性。为确定 siRNA-GC-PEI 纳米粒子在体内特异靶向肿瘤的能力，将 Cy5.5 标记的含 siRNA 的纳米粒子注射入携带 SCC7 肿瘤的小鼠体内，经过近红外荧光信号监测。Cy5.5-siRNA-GC-PEI 纳米粒子在注射后的 1 小时内在肿瘤组织中显示出强烈的近红外荧光信号，并信号持续至两天，表明纳米粒子具有较高的肿瘤靶向能力。此外，由于高肿瘤靶向能力，纳米粒子显著抑制了 B16F10-RFP 肿瘤携带小鼠的 RFP 基因表达。

1. Download : Download high-res image (805KB)
   下载：下载高分辨率图片（805KB）
2. Download : Download full-size image
   下载：下载全尺寸图像

Figure 2. (A) Schematic diagram of siRNA encapsulated hydrophobically modified glycol chitosan and PEI nanoparticles (siRNAGC-PEI NP). (B) Upper; in vivo NIR fluorescence imaging of SCC7 tumor-bearing mice after 1 h post-injection of Cy5.5-siRNA and Cy5.5-siRNA-GC-PEI NPs, lower; ex vivo imaging of the excised SCC7 tumor after 24 h post-injection. (C) In vivo reduction of targeted proteins by siRNA and siRNA-GC-PEI NPs after intravenous injection in B16F10-RFP tumor-bearing mice. The mice were observed by light microscopy and by NIR fluorescence imaging system.
图 2. (A) 结构示意图，显示了被包裹在疏水修饰的聚乙烯亚胺和聚乙二醇基壳寡糖(siRNA-GC-PEI NP)纳米粒子中的 siRNA。 (B) 上：体内近红外荧光成像显示 SCC7 肿瘤携带小鼠在注射 Cy5.5-siRNA 和 Cy5.5-siRNA-GC-PEI NP 后 1 小时的成像，下：在注射后 24 小时，切除的 SCC7 肿瘤的体外成像。 (C) 体内通过靶向蛋白质在 B16F10-RFP 肿瘤携带小鼠经静脉注射 siRNA 和 siRNA-GC-PEI NP 后的变化。小鼠通过光学显微镜和近红外荧光成像系统进行观察。

Adapted with permission from ref. [36].
经过 36 号文献许可后调整。

Modern polymer chemistry and imaging science have created new strategies in the design of polymer-based gene carriers that efficiently deliver genes to targeted regions and image the therapeutic efficacy. The importance of polymeric biomaterials will continue to grow and lead to the development of sophisticated nanosized vectors for gene delivery and imaging and specifically to monitor therapeutic efficacy.
现代聚合物化学和成像科学为设计高效传递基因至靶向区域并成像治疗效果的基于聚合物的基因载体开创了新的策略。聚合物生物材料的重要性将不断增长，并且将推动先进的纳米载体的发展，用于基因传递和成像，特别是用于监测治疗效果。

Dendrimer-based nanoparticles
基于树枝状高分子的纳米粒子

Dendrimers are multivalent macromolecules based on a well-defined cascade motif with spherical shapes, defect-free and perfectly monodisperse characteristics. A typical dendrimer contains three distinct components: a central core, repeated branches and terminal functional groups. The highly controlled structural characteristics of dendrimers have made them useful in biomedicine, for example, as drug/gene delivery systems, catalytic agents, immuno-diagnostics, and contrast agents [37], [38], [39]. Using dendrimers for gene delivery has had a long and contentious past [40]. The amino-terminated PAMAMs are the most frequently used dendrimers for gene transfection. Electrostatic interactions between the anionic phosphate groups of the DNA backbone and the positively charged PAMAM dendrimers result in the formation of nanoscale complexes that prevent the degradation of DNA. Generally, the generation (G) and peripheral structure of dendrimers are significant in gene delivery. Low G of dendrimers result in weak electrostatic interactions between the dendrimers and DNA leading to low transfection efficiency. High G dendrimers display high DNA condensation ratio for better transfection but biocompatibility is decreased because a large number of cationic amino groups are introduced on the dendrimers. Therefore, an appropriate balance between transfection efficiency and toxicity must be met for dendrimer-based gene deliveries. To date, several types of dendrimer-based NP systems are being developed for gene delivery and monitoring gene therapy.
树状大分子是基于明确级联结构的多价大分子，具有球形形态、无缺陷和完全单分散特性。典型的树状大分子包括三个不同组分：中心核心、重复支链和末端功能基团。树状大分子的高度可控制的结构特性使其在生物医学领域具有多种用途，例如作为药物/基因传递系统、催化剂、免疫诊断和造影剂。使用树状大分子进行基因传递已经有很长且争议的历史。氨基端 PAMAMs 是最常用于基因转染的树状大分子。DNA 骨架的阴离子磷酸基团与阳离子性质的 PAMAM 树状大分子之间的静电相互作用导致纳米级复合物的形成，防止 DNA 的降解。通常，树状大分子的代数（G）和外围结构对基因传递至关重要。低代数的树状大分子导致树状大分子和 DNA 之间的静电相互作用较弱，导致传递效率低。高代数的树状大分子显示出较高的 DNA 浓缩比，从而增加传递效率，但生物相容性下降，因为引入了大量阳离子氨基团。因此，必须在基于树状大分子的基因传递中找到传递效率和毒性之间的适当平衡。到目前为止，已经开发了几种基于树状大分子的纳米粒子系统，用于基因传递和监测基因治疗。

Navarro et al. demonstrated that a combination of well-tolerated PAMAM gene delivery carrier with an optimized expression plasmid can result in long-lasting transgene expression in tumor tissue after intravenous vector injection [41]. Polyplexes consisting of a pCMV-Luc (CMV promoter driven luciferase plasmid) condensed with PAMAM G4 and G5 efficiently protected DNA from enzymatic degradation and transfected tumor cells in vitro. Intravenous administration of pCpG-hCMV-Luc/PAMAM polyplexes into Neuro2A neuroblastoma tumor-bearing A/J mice showed predominant luciferase reporter gene expression in the tumor and its transfection efficiency was confirmed by bioluminescence imaging. In contrast, low-molecular-weight PEI (LPEI) polyplexes led to high gene expression in the lung and low luminescence signal in the tumor area. To monitor in vivo biodistribution, each polyplex was fluorescently labeled with near-infrared (NIR) quantum dots (quantoplexes) and revealed lung accumulation for both PAMAM and LPEI-based formulations, demonstrating that biodistribution and transgene expression of polyplexes do not necessarily correlate with each other.
Navarro 等人表明，PAMAM 基因传递载体与优化表达质粒的组合可导致靶向靶向瘤组织的长期基因表达。PAMAM G4 和 G5 结合 pCMV-Luc（CMV 启动子驱动的荧光素酶质粒）形成的聚合物复合物有效保护 DNA 免受酶降解，并在体外转染了肿瘤细胞。将 pCpG-hCMV-Luc/PAMAM 复合物经静脉注射到带有 Neuro2A 神经母细胞瘤的 A/J 小鼠中，在肿瘤中显示出主要的荧光素报告基因表达，转染效率通过生物发光成像得到证实。相比之下，低分子量 PEI（LPEI）复合物导致肺部基因表达高，而肿瘤区域发光信号低。为了监测体内生物分布，每种复合物均被近红外（NIR）量子点荧光标记（量子复合物），显示出基于 PAMAM 和 LPEI 的配方均在肺部聚集，表明复合物的分布和基因表达不一定相互相关。

In another report, a targeting ligand modified PAMAM has been designed and evaluated for efficient brain-targeting gene delivery [42]. A 29 amino acid peptide derived from the rabies virus glycoprotein (RVG29) was modified on PAMAM G5 through bifunctional PEG and complexed with the plasmid, yielding PAMAM-PEG-RVG29/DNA NPs. RVG29 is known to bind to the acetylcholine receptor (AchR) which is widely expressed in the brain including capillary endothelial cells [43]. PAMAM-PEG-RVG29/DNA showed higher blood–brain barrier (BBB)-crossing and gene expression efficiency than PAMAM/DNA NPs in an in vitro BBB model and in a small animal model. In vivo distribution of NPs was evaluated after systemic injection of fluorophore-labeled PAMAM-PEG-RVG29/DNA and NPs were preferably accumulated in the brain as compared with NPs without a targeting peptide.
在另一份报告中，设计并评估了用于有效脑靶向基因传递的靶向配体修饰 PAMAM [42]。从狂犬病病毒糖蛋白(RVG29)中衍生的一个 29 氨基酸肽被通过双官能 PEG 修饰在 PAMAM G5 上，并与质粒形成 PAMAM-PEG-RVG29/DNA NPs。RVG29 被认为能与广泛表达于脑内的乙酰胆碱受体(AchR)结合[43]。在体外 BBB 模型和小动物模型中，PAMAM-PEG-RVG29/DNA 显示比 PAMAM/DNA NPs 更高的穿越血脑屏障(BBB)和基因表达效率。通过尾静脉注射标记荧光素的 PAMAM-PEG-RVG29/DNA，评估了 NPs 的体内分布，与无靶向肽的 NPs 相比，NPs 更多地积聚在大脑中。

Different types of dendrimers were also used for gene delivery and imaging. Polyglycerol (PG)-based dendrimer core shell structures exhibiting low cytotoxicity have been developed to delivery siRNA to tumors in vivo [44]. In this study, different PG- and PEI-derived, high molecular weight, dendritic structures were synthesized by combining PG, PEI, glucose and pentaethylenehexamine (PEHA). In vitro, PG-amine (aminated PG) and PEI–Glu (glucose conjugated PEI)-based luciferase siRNA-polyplexes successfully accumulated in the cytoplasm in a time-dependent manner and decreased bioluminescence in luciferase-expressing glioblastoma cells (U87-Luc) and in primary endothelial cells. PG-amine exhibited the best ratio of silencing efficacy and enhanced siRNA transfection efficiency with reduced toxicity compared with other dendritic systems examined. Significant gene silencing of 50% was accomplished in vivo within 24 h of siRNA-PG-amine polyplexes treatment. In vivo silencing of the luciferase gene by siRNA-PG-amine and delivery of siRNA into tumors were verified by three different ways: (1) bioluminescent imaging in SCID mice bearing U87-Luc tumors, (2) bioluminescent/fluorescent imaging in BALB/C mice bearing DA3-mCherry-Luc tumors, and (3) microscopy imaging of FITC-labeled siRNA in tumor tissues.
不同类型的树状高聚物也被用于基因传递和成像。基于聚甘油（PG）的树状高聚物核壳结构展示出较低的细胞毒性，已被开发用于体内向肿瘤传递 siRNA[44]。在这项研究中，通过结合 PG、PEI、葡萄糖和五乙二胺六次胺（PEHA），合成了不同的 PG-和 PEI 衍生的高分子量树状结构。在体外实验中，PG-氨基（氨基化 PG）和 PEI-Glu（葡萄糖共轭的 PEI）基的荧光素酶 siRNA-聚合物复合物按时间顺序成功地在细胞质中积聚，并降低了表达荧光素酶的胶质母细胞瘤细胞(U87-Luc)和原代内皮细胞的生物发光。 与其他树状系统相比，PG-氨基具有最佳的沉默效能比和增强的 siRNA 转染效率，并减少了毒性。在 siRNA-PG-胺聚合物复合物处理后的 24 小时内，在体内成功实现了 50%的基因沉默。通过三种不同方式验证了 siRNA-PG-胺对荧光素酶基因的体内沉黙和对肿瘤中 siRNA 的传递：（1）在携带 U87-Luc 肿瘤的 SCID 小鼠中进行生物发光成像，（2）在携带 DA3-mCherry-Luc 肿瘤的 BALB/C 小鼠中进行生物发光/荧光成像，（3）荧光素标记的 siRNA 在肿瘤组织中的显微成像。

Chisholm et al. reported NPs composed of 3G polypropylenimine dendrimers (PPIG3) that are capable of tumor transfection upon systemic administration in tumor-bearing mice [45]. These NPs were prepared by complexing the PPIG3 dendrimer with a plasmid DNA encoding sodium iodide symporter (NIS) to form colloidal and stable self-assembled NPs with sizes ranging from 30 to 300 nm depending on the DNA concentration. Specific gene transfection efficiency was imaged by using small-animal nano-single-photon emission computed tomography/computer tomography (nanoSPECT/CT) scanner. Using nuclear whole-body imaging and NIS as a reporter gene, they were able to detect a specific and unique radiotracer uptake in tumors of both immunodeficient and immunocompetent mice whereas no signal was detected in normal animal tissue.
Chisholm 等报道了由 3G 聚丙烯胺树状聚合物（PPIG3）组成的纳米粒子，在肿瘤携带小鼠体内的系统给药后能够实现转染。这些纳米粒子是通过将 PPIG3 树状聚合物与编码钠碘转运体（NIS）的质粒 DNA 复合而制备的，形成胶体稳定的自组装纳米粒子，大小在 30 至 300nm 之间，取决于 DNA 浓度。利用小动物纳米单光子发射计算机断层扫描（nanoSPECT/CT）扫描仪来成像特定基因转染效率。通过核全身成像和 NIS 作为记者基因，他们能够检测到免疫缺陷和免疫能力小鼠肿瘤中特定和独特的放射性示踪剂摄取，而在正常动物组织中没有检测到信号。

Dendrimers are expected to play a key role in biomedical fields in the future as they have shown efficient gene delivery when the ratio of genetic material and dendrimer is achieved. Advances in understanding the role of molecular weight and architecture on the function of dendrimers together with recent progress in molecular imaging will enable the application of these branched polymers as molecular imaging probes for gene therapy. Recently, various types of dendrimer-based inorganic NPs were also developed and this will be discussed in the following sections.
树状分子在生物医学领域的未来中预计将发挥关键作用，因为它们在遗传物质和树状分子比例达到时展现了有效的基因传递。对树状分子的分子量和结构对功能的了解日益增加，再加上分子成像的最新进展，将使这些分支聚合物能够作为基因治疗的分子成像探针应用。最近，还开发了各种类型的基于树枝聚合物的无机纳米粒子，这将在接下来的章节中进行讨论。

Lipid-based nanoparticles
基于脂质的纳米颗粒

Lipid-based NPs, such as liposomes or micelles, have been used extensively in the past few decades as gene delivery vehicles [46], [47], [48]. Generally, lipid-based NPs interact with negatively charged nucleic acids through electrostatic interactions to form lipoplexes. Many lipid-based gene delivery approaches are currently being tested at the clinical level [46]. Additionally, the lipid coating also ensures good pharmacokinetics and an improved biocompatibility of the NPs for biomedical applications [49]. A relatively new and promising application of lipid-based NPs is their use for molecular imaging of gene delivery [50], [51]. The multifunctional character of lipid NP platforms has significant advantages because it allows for the inclusion of a variety of imaging agents ranging from fluorescent molecules to nanocrystals, including QDs, iron oxide NPs, and gold NPs [50], [52], [53]. These carriers can also be surface modified to carry a variety of other compounds such as targeting peptides by incorporating into or binding to the cationic phospholipids membrane [54], [55]. Their use as gene delivery vehicles is made possible by the positively charged surface that can adsorb negatively charged genes [56].
过去几十年中，基于脂质的纳米粒子，如脂质体或胶束，作为基因传递载体得到广泛应用。通常，脂质基纳米粒子通过静电相互作用与带负电荷的核酸结合形成脂质体复合物。目前有许多基于脂质的基因传递方法正在临床级别测试中。此外，脂质包被还确保了纳米粒子在生物医学应用中的良好药代动力学和改进的生物相容性。脂质基纳米粒子相对较新且有前景的应用是它们用于基因传递的分子成像。脂质纳米粒子平台的多功能性具有显着优势，因为它允许包含各种成像试剂，从荧光分子到纳米晶体，包括量子点、氧化铁纳米粒子和金纳米粒子等。这些载体也可以表面改性以携带各种其他化合物，如通过纳入或结合到阳离子磷脂膜的作用，携带靶向肽等。它们作为基因传递载体的应用是通过可以吸附带负电荷基因的正电荷表面实现的。

Yagi et al. described a systemically injectable siRNA vehicle, the wrapsome (WS), which contains siRNA and a cationic lipofection complex in a core that is fully enveloped by a neutral lipid bilayer and hydrophilic polymer, PEG [57]. These NPs physically contain the siRNA and protect it from degradation and rapid dissociation from the particle. WS particles were prepared as 100 nm in diameter to maximize the enhanced permeation retention (EPR) effect [58]. In vivo efficacy of WS was tested by using KLF5-siRNA, which is known to play a role in tumor angiogenesis. KLF5-siRNA/WS exhibited significant antitumor activity; although, neither WS containing control scrambled-siRNA nor saline containing KLF5-siRNA affected tumor growth. KLF5-siRNA/WS inhibited KLF5 expression within tumors at both mRNA and protein levels without acute or long-term toxicity. Furthermore, NIR whole-body imaging was used to analyze the distribution of siRNA after systemic administration of Cy5-labeled siRNA/WS which confirmed its tumor specificity in vivo and ex vivo. Another form of lipid-based NPs were developed as siRNA delivery carriers based on the use of a new cationic lipid, N,N″-dioleyglutamide (DG). DG was synthesized by peptide bond linkage of oleylamine to each carboxylic acid group of glutamic acid. DG-based cationic liposomes and siRNA form stable complexes and show high siRNA transfection efficiency and low cytotoxicity compared with conventional cationic lipid-based liposomes and commercially available Lipofectamine 2000. In vivo reduction of target proteins by siRNA complexed to DG was visualized by fluorescence imaging in mice bearing B16F10-RFP tumors.
Yagi 等人描述了一种全身注射可行的 siRNA 载体，包含 siRNA 和阳离子脂质体复合物的包裹体（WS），其核心完全被中性脂质双层和亲水聚合物 PEG 所包裹[57]。这些 NP 物理上包含 siRNA 并保护它免受降解和迅速从颗粒中解离。WS 颗粒直径为 100nm，以最大程度地增强渗透保留（EPR）效应[58]。通过使用已知在肿瘤血管生成中发挥作用的 KLF5-siRNA 测试了 WS 的体内有效性。 KLF5-siRNA/WS 表现出显著的抗肿瘤活性；虽然既 WS 含有对照乱序 siRNA，也含有 KLF5-siRNA 的盐水并未影响肿瘤生长。KLF5-siRNA/WS 抑制了肿瘤内 KLF5 的 mRNA 和蛋白表达，没有急性或长期毒性。此外，通过 NIR 全身成像分析了 Cy5 标记的 siRNA/WS 全身给药后的 siRNA 分布，证实了其在体内和体外的肿瘤特异性。另一种基于脂质的 NP 形式被开发为 siRNA 传递载体，基于使用新的阳离子脂质 N，N″-二油酰谷氨酰胺（DG）。通过将油胺基与谷氨酸的每个羧酸基团进行肽键连接来合成 DG。基于 DG 的阳离子脂质体和 siRNA 形成稳定的复合物，并且与传统的阳离子脂质体和商业可获取的 Lipofectamine 2000 相比，显示出高 siRNA 转染效率和低细胞毒性。通过荧光成像在携带 B16F10-RFP 肿瘤的小鼠中观察到，通过与 DG 形成复合物的靶蛋白的体内减少。

Ukawa et al. developed multifunctional lipid envelope-type nanoparticles for gene delivery in which plasmid DNA/siRNA is condensed with a polycation, followed by encapsulation by a lipid envelope [59], [60]. In this design, pH-sensitive fusogenic peptide (GALA) was introduced into liposomal membranes using a cholesteryl moiety for anchoring. The intracellular trafficking of dissociation, as well as the endosome escape process of the nanoformula after receptor-mediated endocytosis was successfully revealed by a quantitative imaging analysis involving nuclear isolation, real-time PCR, and confocal laser scanning microscopy. The incorporation of cholesteryl GALA (chol-GALA) into the lipid envelope enhanced membrane fusion with the endosome, and then increased the transfection activity and gene knockdown activity of the encapsulated plasmid DNA or siRNA. Interestingly, additional coating of GALA-modified liposomes with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer resulted in enhanced transfection activity of encapsulating plasmid DNA in primary hepatocytes [60]. The presence of an MPC polymer-coating supported cellular uptake and the subsequent cytoplasmic dissociation of DNA from the lipid envelope by assisting the endosome-fusogenic function of GALA. Such MPC/GALA-modified lipid nanoparticles appear to be a great potent carrier for a liver-targeting gene delivery system.
Ukawa 等人开发了多功能脂质包裹型纳米颗粒，用于基因传递，其中质粒 DNA/siRNA 与多聚阳离子缩合，随后被脂质包裹所封装。在这个设计中，pH 敏感的促融合肽（GALA）通过胆固醇基团用于定位，在脂质体膜中引入。定量成像分析揭示了受体介导的内吞作用后，纳米配方的细胞内分离和内体逃逸过程，包括核分离、实时 PCR 和共聚焦激光扫描显微镜检查。胆固醇 GALA（chol-GALA）的引入增强了脂质包裹体与内体的融合，从而增加了封装的质粒 DNA 或 siRNA 的转染活性和基因沉默活性。有趣的是，将 GALA 修饰的脂质体额外包覆 2-甲基丙烯酰氧基乙基磷酸胆碱（MPC）聚合物，结果是提高了原代肝细胞中封装质粒 DNA 的转染活性。MPC 聚合物包覆的存在支持了细胞摄取以及通过协助 GALA 内体融合功能进行的 DNA 与脂质包裹的细胞质分离。这种 MPC/GALA 修饰的脂质纳米颗粒似乎是一个适用于肝靶向基因传递系统的优秀载体。

The relative ease of preparation, the flexibility, and most importantly, the biocompatibility of lipid-based NPs make them useful tools as multifunctional NP. Recently, lipid-based inorganic NPs were developed as siRNA gene carriers and imaging agents. Oliver et al. reported a novel liposome formulation containing a lipidic gadolinium contrast agent [61]. The gadolinium liposome was found to be an effective vehicle for transport of plasmid DNA into cells, which shows a promising application for molecular imaging of gene therapy. The relative ease of preparation, the flexibility and, most importantly, the biocompatibility of lipid-based NPs make them useful tools as multifunctional NP. Ongoing developments in bionanotechnology may lead to the development of lipid-based NPs for molecular imaging of gene delivery with better imaging properties, improved stability and a more defined size and structure.
脂质基纳米粒子（NPs）具有制备简便、灵活性和生物相容性，使其成为多功能 NPs 的有用工具。最近，脂质基无机 NPs 已被开发为 siRNA 基因载体和成像剂。奥利弗等报道了含脂质钆对比剂的新型脂质体配方。发现钆脂质体对于携带质粒 DNA 进入细胞是一种有效载体，展示了在基因治疗的分子成像中具有很有前景的应用。脂质基 NPs 的制备简便、灵活性以及生物相容性使其成为多功能 NPs 的有用工具。生物纳米技术的持续发展可能导致脂质基 NPs 的发展，以用于基因传递的分子成像，具有更好的成像性能、改善的稳定性以及更明确定义的大小和结构。

Iron oxide nanoparticles 氧化铁纳米颗粒

Iron oxide NPs (IONPs) have a long history of investigation and have shown remarkable potentials in biomedical research, including MRI contrast enhancement, drug delivery, hyperthermia, and cell separation/labeling [62], [63], [64], [65], [66], [67], [68]. The popularity of IONPs is mainly because they: (1) provide an MR-based read-out, in particular on T₂^*-weighted images; (2) can be magnetically manipulated and change their magnetic properties; (3) can be biologically degraded, metabolized and integrated into serum Fe pool to form hemoglobin or to enter other metabolic processes; (4) have a large surface area for carrying drugs and genes. These features give IONPs great advantages for in vivo molecular imaging and drug/gene delivery.
纳米级氧化铁颗粒（IONPs）在生物医学研究中有着悠久的历史，并展现出在 MRI 对比增强、药物输送、热疗以及细胞分离/标记方面的显著潜力 [62], [63], [64], [65], [66], [67], [68]。IONPs 的普及主要是因为它们：（1）提供基于 MR 的读出，特别是在 T ₂ ^* 加权图像上；（2）可以被磁性操纵并改变它们的磁性能；（3）可以在生物体内被降解、代谢并整合到血清铁库中形成血红蛋白或参与其他代谢过程；（4）具有用于携带药物和基因的大表面积。这些特点使得 IONPs 在体内分子成像和药物/基因传递方面具有极大优势。

Like other inorganic NPs, surface modification processes are necessary to stabilize IONPs for strong binding enhancement of the therapeutic gene and to control the release mechanism. Without optimized surface modification the gene may not strongly bind to IONPs, resulting in the instant release of the therapeutic gene during the delivery. Positive charges are the most suitable surface charges for IONP applications in gene delivery. Thus, a large amount of negatively charged DNA molecules can bind on the positively charged surface of the particles by utilizing electrostatic interactions. A popular choice for this approach is to use cationic polymers such as PEI, PLL, and chitosans. Various PEI-coated IONPs were reported for in vitro magnetic NP-mediated non-viral gene delivery [69], [70]. However, the in vivo applications of these NPs have been limited due to concerns over cellular toxicity.
与其他无机颗粒一样，表面修饰过程对于稳定 IONPs 以增强对治疗基因的结合并控制释放机制至关重要。在没有经过优化表面修饰的情况下，基因可能无法牢固结合到 IONPs 上，导致治疗基因在传递过程中瞬间释放。正电荷是 IONP 应用于基因传递中最适合的表面电荷。因此，通过静电相互作用，大量带负电荷的 DNA 分子可以结合到粒子的正电荷表面上。采用这种方法的一个热门选择是使用阳离子聚合物，如 PEI、PLL 和壳聚糖。报道了各种 PEI 包被的 IONPs 用于体外磁性 NP 介导的非病毒基因传递 [69], [70]。然而，由于细胞毒性的担忧，这些 NP 的体内应用受到限制。

To overcome these limitations, various polymers have been used to coat or conjugate to the IONPs. PEGylated PEI has been synthesized and applied to IONPs for targeted gene delivery and imaging. Chen et al. reported a T cell specific ligand, the CD3 single chain antibody (scAb_CD3), conjugated to PEG-g–PEI stabilized IONPs for gene delivery to T cells for immunosuppression [71]. Based on a reporter gene assay, scAb_CD3–PEG-g–PEI functionalized IONPs led to 16-fold gene transfection enhancement in rat T lymphocyte HB8521 cells with low cytotoxicity, demonstrating effective T-lymphocyte-targeted immunotherapy. Furthermore, this targeting process in cells was successfully imaged by MRI. Self-assembled chitosan was also used as an effective coating to stabilize IONPs. A hepatocyte-targeted gene delivery and imaging system has been developed by using ^99mTc-labeled IONPs-loaded chitosan-linoleic acid NPs (^99mTc-SCLNs) [72]. After intravenous injection of NPs, SCLNs were specifically driven to the hepatocytes by linoleic acid, which accumulate in hepatocytes and play a central role in the liver. Furthermore, SCLN complexes containing enhanced green fluorescence protein (pEGFP) plasmid expressed GFP in hepatocytes after administration in mice and the selective accumulation of NPs in hepatocytes could be monitored. Recently, Kievit et al. engineered an novel copolymer, CP-PEI, to coat onto IONPs by combining the biocompatibility of chitosan and the steric stabilization of PEG with the large positive charge of PEI [73]. The NP-CP-PEI was designed for stable binding, protecting, and delivering of DNA for gene expression while maintaining IONP properties and high biocompatibility. In vivo, the NP-CP-PEI demonstrated a high level of GFP expression in a C6 xenograft mouse model and showed enhanced MR contrast in cells.
为了克服这些局限性，已经使用各种高分子材料来包覆或共轭到 IONPs 上。合成了 PEG 基 PEI 并应用于 IONPs，用于靶向基因传递和成像。Chen 等人报告了一种 T 细胞特异性配体，CD3 单链抗体(scAb)，与 PEG-g-PEI 稳定的 IONPs 结合，用于 T 细胞免疫抑制的基因传递。根据记者基因分析，scAb-PEG-g-PEI 功能化的 IONPs 导致大鼠 T 淋巴细胞 HB8521 细胞中基因转染增强 16 倍，具有低细胞毒性，表明了有效的 T 淋巴细胞靶向免疫治疗。此外，通过 MRI 成功对细胞中的靶向过程进行了成像。自组装的壳聚糖也被用作稳定 IONPs 的有效包衣。通过使用 Tc 标记的 IONPs 载荷的壳聚糖-亚油酸纳米粒(SCLNs)，已经开发了一个以肝细胞为靶向的基因传递和成像系统。在 NPs 静脉注射后，通过亚油酸将 SCLNs 特异性驱动到肝细胞，亚油酸在肝细胞中积聚并在肝脏中起核心作用。此外，将表达增强绿色荧光蛋白质（pEGFP）质粒的 SCLN 复合物注入小鼠后，肝细胞中表达 GFP，并且可以监测 NPs 在肝细胞中的选择性积聚。最近，Kievit 等人设计了一种新型共聚物 CP-PEI，通过结合壳聚糖的生物相容性、PEG 的立体稳定性以及 PEI 的大正电荷，将其包覆到 IONPs 上。NP-CP-PEI 被设计用于稳定结合、保护和传递 DNA 以进行基因表达，同时保持 IONP 的性能和高生物兼容性。 在体内实验中，NP-CP-PEI 在 C6 异种移植小鼠模型中显示出高水平的 GFP 表达，并在细胞中展示了增强的 MR 对比度。

Multifunctional probes based on IONPs for multimodality imaging and delivery of genes have been developed as well [19], [74], [75]. Noninvasive dual-modality imaging was accomplished using multifunctional IONPs for simultaneous in vivo imaging and transfer of siRNAs into tumors by both MRI and fluorescence optical imaging [19]. The resulting probe consisted of aminated dextran-coated IONPs labeled with Cy5.5 and two different linkers: membrane translocation peptides (MPAP, for intracellular delivery) and siRNA molecule targeting GFP (siGFP) (Fig. 3). Tracking results in vivo of these multifunctional IONPs by MR and NIR fluorescence imaging demonstrated that they could simultaneously deliver siRNAs and monitor therapeutic efficacy.
基于 IONPs 的多功能探针已被开发用于多模态成像和基因传递[19], [74], [75]。利用多功能 IONPs 实现了非侵入性双模态成像，可以同时进行体内成像并通过 MRI 和荧光光学成像将 siRNA 转移到肿瘤中[19]。结果显示，该探针由氨基葡聚糖包被的 IONPs 标记为 Cy5.5，具有两种不同的连接物：膜转位肽（MPAP，用于细胞内传递）和靶向 GFP 的 siRNA 分子（siGFP）（图 3）。利用 MR 和 NIR 荧光成像对这些多功能 IONPs 在体内的追踪结果表明，它们可以同时传递 siRNA 并监测治疗效果。

1. Download : Download high-res image (330KB)
   下载：下载高分辨率图像（330KB）
2. Download : Download full-size image
   下载：下载全尺寸图像

Figure 3. Functional iron oxide nanoparticles (IONPs) for molecular imaging of gene delivery. (A) Schematic illustration of the multifunctional nanoparticles consisting of a magnetic nanoparticle labeled with Cy5.5, membrane translocation peptides (MPAP), and short-interfering ribose nucleic acid (siRNA) molecules. (B) In vivo magnetic resonance imaging (MRI) of mice bearing subcutaneous tumors before and after treatment. High-intensity optical signal in the tumor confirmed the delivery of the nanoparticle.
图 3. 用于基因导入分子成像的功能性氧化铁纳米粒子（IONPs）。（A）多功能纳米粒子示意图，包括标记有 Cy5.5 的磁性纳米粒子，膜转位肽（MPAP）和短干扰核糖核酸（siRNA）分子。（B）治疗前后携带皮下肿瘤的小鼠的体内磁共振成像（MRI）。肿瘤中的高强度光学信号证实了纳米粒子的导入。

Adapted with permission from ref. [19].
经过许可后改编自参考文献[19]。

In another example oligodeoxynucleotide (ODN) was attached to the surface of the IONPs by employing cleavable linkers [76], [77]. In this study, a phosphorothioate-modified ODN (sODN) complementary to the target mRNA was labeled with rhodamine or FITC on the 5′-end and biotin on the 3′-end, allowing the biotinylated ODN to conjugate to IONPs via an avidin–biotin linkage. NPs were delivered by intracerebroventricular infusion to the cerebral ventricle of mice. Superparamagnetic iron oxide (SPIO) nanoparticles with sODN in the living brain could be imaged with MRI. Another design of IONPs multifunctional imaging and gene delivery was reported by Agrawal et al. In this study, dendrimer-conjugated magnetofluorescent nanoworms (dendriworms) as carriers and imaging agents for siRNA delivery were engineered [78]. To prepare dendriworms, cysteamine core PAMAM G4 dendrimers were reduced and conjugated via a heterobifunctional linker to cross-linked iron oxide nanoworms which were amine-modified and NIR dye labeled. Dendriworms were able to delivery cargo into cells with negligible toxic effects on cells and delivered siRNA 2.5-fold more efficiently than commercial cationic lipids. In addition, anti-EGFR dendriworms led to specific and significant suppression of EGFR expression in an EGFR-driven transgenic model of glioblastoma and enabled fluorescent tracking of siRNA delivery in vivo.
另一例中，通过使用可切断的连接剂将寡脱氧核苷酸（ODN）连接到离子氧化铁纳米粒子（IONPs）表面的研究中，对目标 mRNA 的磷酸硫酸酯修饰的 ODN（sODN）在 5'-末端标记有罗丹明或荧光异吲哚啉（FITC），在 3'-末端标记有生物素，从而使生物素化的 ODN 能够通过亲和素-生物素连接结构结合到 IONPs 上。纳米颗粒通过侧脑室注射送达到小鼠脑室。在 MRI 下，具有 sODN 的超顺磁氧化铁（SPIO）纳米颗粒可以在活体大脑中成像。Agrawal 等人报道了 IONPs 多功能成像和基因传递的另一设计。该研究中，以树状分子为载体和成像剂的磁荧光纳米蠕虫（dendriworms）用于 siRNA 传递。为制备 dendriworms，将半胱氨酸核 PAMAM G4 树状分子通过异双功能连接剂还原和结合到氨基修饰和近红外荧光染料标记的交联氧化铁纳米蠕虫上。Dendriworms 能够将载体传递入细胞，对细胞几乎没有毒性影响，并且比商业阳离子脂质效率高 2.5 倍。此外，抗 EGFR 树状蠕虫导致 EGFR 驱动的胶质母细胞瘤转基因模型中 EGFR 表达的特异性和显著抑制，并且能够在体内对 siRNA 传递进行荧光追踪。

Quantum dots 量子点

Quantum dots (QDs) are colloidal nanosized semiconductor particles. These NPs can be excited over a wide range of wavelengths and emit specific sharp bands with limited photobleaching that can be fine-tuned during the synthesis technique [79], [80]. In addition to the advantages that these NPs have for molecular imaging, QDs can also be used as intracellular carriers for various biomolecules such as proteins, antibodies, and genes. QDs have been used extensively as cell labels, tissue imaging agents, FRET donors, and sensing agents. The wide variety of QD applications in the biomedical field has been summarized elsewhere [81], [82], [83], [84], [85].
量子点（QDs）是胶体纳米半导体颗粒。这些纳米颗粒能够在广泛波长范围内被激发，并发射特定的尖锐波段，具有有限的光漂白，可以在合成技术中进行细致调节。除了这些纳米颗粒在分子成像方面的优势外，QDs 还可以作为细胞内载体，用于携带各种生物分子，如蛋白质，抗体和基因。QDs 被广泛应用于细胞标记、组织成像试剂、FRET 给体和传感剂。在生物医学领域中，QD 应用的广泛种类已经在其他地方总结[81]，[82]，[83]，[84]，[85]。

To improve the knockdown efficacy, various delivery techniques are used to deliver QDs into the nucleus. Srinivasan et al. designed QD-decorated plasmid DNA for long-term intracellular and intranuclear tracking studies [86]. Numerous phospholipid-coated, CdSe/ZnS core/shell QDs were conjugated to plasmid DNA via a peptide nucleic acid (PNA)-N-succinimidyl-3-(2-pyridylthio) propionate linker. These QD–DNA conjugates were capable of expressing enhanced green fluorescent protein in the presence of Lipofectamine 2000 and monitored by fluorescence microscopy, while the cellular uptake of QD–DNA conjugates was monitored in real-time at the same time. Overall, transfection was 62% more efficient than unconjugated plasmid DNA.
为提高击倒效果，使用各种传递技术将量子点(QDs)送入细胞核。Srinivasan 等设计了 QD 修饰质粒 DNA，用于长期细胞内和核内跟踪研究。大量磷脂质涂层的 CdSe/ZnS 核/壳结构 QDs 通过肽核酸(PNA)-N-琥德基亚胺基-3-(2-吡啶硫)丙酸酯连接剂与质粒 DNA 结合。这些 QD-DNA 共轭物在存在 Lipofectamine 2000 的情况下可以表达增强型绿色荧光蛋白，并通过荧光显微镜进行监测，同时可实时监测细胞对 QD-DNA 共轭物的摄取。总体上，转染效率比未结合的质粒 DNA 高 62%。

Other functionalizing techniques have been used to improve cell and nucleus internalization. By using a PEGylated QD core as a scaffold, siRNA and a FITC-labeled tumor targeting peptide FITC-F3 were conjugated to the particle surface [87]. F3 peptide is a 31 amino acid peptide that has been shown to target tumor cells in vitro and in tumor-bearing mice when systemically administered as a free peptide or conjugated with PEGylated QDs [88]. In an EGFR model system, prepared F3/siRNA–QDs produced significant knockdown of EGFR signal (29%) after delivery to cells and release of siRNA from their endosomal entrapment [87]. Delivery of siRNA containing QDs was successfully imaged and tracked by fluorescent microscopy. Recent works have focused on preventing the use of additional chemical treatments for cellular internalization to minimize its toxicity. Tan et al. showed that QDs can be encapsulated into polymer NPs and used as self-tracking carriers for human epidermal growth factor receptor 2 (HER-2) siRNA [89]. When positively charged chitosan NPs are used in conjunction with the fluorescent probes, the particles are internalized into cells, gene delivery is easily monitored in vitro, and the HER-2 gene is successfully silenced [89]. Another form of polymer encapsulated QDs were developed for effective and safe siRNA delivery via a proton-absorbing polymeric coating (proton sponges) [90]. With a balanced composition of positively and negatively charged functional groups on the QD surface, such as carboxylic acid and tertiary amine, these NPs can be designed to specifically release the trapped siRNAs into the cytoplasm. Proton-sponge layer coated QDs allowed for siRNA adsorption and demonstrated a 10–20 fold improvement in gene silencing efficiency. Without the use of Lipofectamine 2000, a six-fold reduction in cellular toxicity was achieved because of the increased rate of endosomal escape. Cellular uptake of these particles could be visualized in real-time due to the fluorescent signal of the QDs while ultrastructural localizations could be determined by electron microscopy by detecting the presence of semiconductors [90].
利用 PEG 化 QD 核作为支架，将 siRNA 和 FITC 标记的肿瘤靶向肽 FITC-F3 偶联到颗粒表面，以提高细胞和细胞核的内在化。F3 肽是一种由 31 个氨基酸组成的肽，已被证明能靶向体外和肿瘤携带小鼠内的肿瘤细胞，当作为自由肽或与 PEG 化 QDs 结合给予系统性给药[88]。在 EGFR 模型系统中，制备的 F3/siRNA-QDs 在递送到细胞后产生显著的 EGFR 信号沉默（29%），从而使 siRNA 从其内体困住中释放[87]。成功实现了含有 QDs 的 siRNA 的递送并通过荧光显微镜进行了成像和跟踪。最近的研究侧重于预防对细胞内部化的额外化学处理以减少其毒性。谭等人表明可以将 QDs 包装到聚合物 NPs 中，并用作自行跟踪载体来转运人表皮生长因子受体 2（HER-2）siRNA[89]。当阳离子化的壳聚糖 NPs 与荧光探针一起使用时，颗粒被内入细胞，基因递送在体外容易监控，并且 HER-2 基因成功被静默[89]。另一种包覆的聚合物 QDs 形式被开发出来，通过具有吸附质子的聚合涂层（质子海绵）实现了对 siRNA 的有效且安全的递送[90]。通过在 QD 表面具有平衡的正电和负电功能基团的组成，如羧酸和三级胺，这些 NPs 可以设计成特定释放被困捕的 siRNAs 到细胞质中。质子海绵层覆盖的 QDs 允许 siRNA 吸附，并显示了基因沉默效率提高了 10-20 倍。 在没有使用 Lipofectamine 2000 的情况下，通过增加逃逸内体的速率，细胞毒性降低了六倍。这些颗粒的细胞摄取可以实时可视化，因为 QDs 的荧光信号，而通过电子显微镜可以检测半导体颗粒的存在，从而确定超微结构的位置。

Fluorescence resonance energy transfer (FRET) is an interaction between the electronically excited states of two fluorophores, in which energy is transferred by long-range dipole-dipole coupling from a donor fluorophore that is in an electronic excited state to an acceptor chromophore. FRET is very sensitive to nanometer-scale changes in donor–acceptor separation distances and their relative dipole orientations, which provides a powerful tool to probe a great variety of biological processes. QDs have size-dependent narrow emission, broad absorption windows and strong resistance to photobleaching, making them well-suited for multi-colored imaging applications. These properties would facilitate effective wavelength separation between donor and acceptor fluorescence and therefore allow the use of QDs as very attractive donors in FRET-based applications. Several comprehensive reviews have summarized their biological applications [91], [92], [93].
荧光共振能量转移（FRET）是两种荧光团的电子激发态之间的相互作用，其中通过远程偶极-偶极耦合从处于电子激发态的供体荧光团向受体色素转移能量。FRET 对供体-受体分离距离和它们的相对偶极取向的纳米级变化非常敏感，这为探测各种生物过程提供了强大的工具。量子点（QDs）具有尺寸相关的窄发射带、宽吸收窗口和强抗光漂白能力，非常适合于多色成像应用。这些特性有助于在供体和受体荧光之间实现有效的波长分离，从而让 QDs 作为 FRET 应用中极具吸引力的供体。一些综合性评述总结了它们的生物应用。

A FRET-based technique was applied to image the release of genetic materials from QDs [94]. QDs were first covalently conjugated with positively charged PEI and then complexed by electrostatic interactions with Cy5-labeled vascular endothelial growth factor siRNA (Cy5-VEGF siRNA) to prepare nanosized PECs (Fig. 4A). A FRET effect occurred between the dye on the VEGF siRNA (the acceptor) and the QD (the donor) when the two components were electrostatically bound and a visible fluorescence overlap was observed. Significantly enhanced delivery was observed with the use of PEI-coated-QDs as opposed to the PEI alone. From confocal imaging analysis, the intraceullar uptake and release of the siRNA was observed during delivery from the different fluorescent signals emitted by the two components during detachment (Fig. 4B and C). The FRET effect not only provided real-time imaging of the gene delivery, but also provided a quantitative evaluation of the siRNA release from the nanoparticle by the use of flow cytometry analysis to monitor when the siRNA and QD were tightly bound and when the two components would start to detach and unpack into the nucleus and hence exhibit two distinct fluorescence spectra (Fig. 4D and E).
通过 FRET 技术对 QDs 释放遗传材料进行成像[94]。首先，QDs 与阳性电荷的 PEI 进行共价共轭，然后与 Cy5 标记的血管内皮生长因子 siRNA（Cy5-VEGF siRNA）通过静电相互作用形成纳米级 PECs（图 4A）。当这两个组分通过静电结合时，VEGF siRNA 上的染料（受体）与 QD（供体）之间发生 FRET 效应，并观察到明显的荧光重叠。使用 PEI 包被的 QDs 相对于单独使用 PEI，观察到显著增强的传递效果。通过共聚焦成像分析，在传递过程中从两个组分在解离过程中发出的不同荧光信号观察到 siRNA 的细胞内摄取和释放（图 4B 和 C）。FRET 效应不仅提供基因传递的实时成像，还通过流式细胞术分析定量评估了 siRNA 从纳米颗粒中的释放，以监测 siRNA 和 QD 何时紧密结合，以及两个组分何时开始解离并释放到细胞核中，并展现出两种不同的荧光光谱（图 4D 和 E）。

1. Download : Download high-res image (748KB)
   下载: 下载高清图片（748KB）
2. Download : Download full-size image
   下载：下载全尺寸图像

Figure 4. (A) Schematic image of the PEI conjugated QDs (QD–PEI) and endocytosis inhibitor conjugated QD–PEI (QD–PEI–Hph-1). Intracellular uptake observed by confocal imaging of (B) Cy5-siRNA to QD–PEI and (C) Cy5-siRNA to QD–PEI–Hph-1. (D) Intensity of FRET signals when QD–PEI is incubated in vitro. (E) The FRET intensity over incubation time as compared to the fluorescence intensity of the Cy5-siRNA alone. Scale bar is 10 μm.
图 4. (A) 显示 PEI 共轭的纳米量子点(QD–PEI)和内吞作用抑制剂共轭的 QD–PEI(QD–PEI–Hph-1)示意图。透过共聚焦显微成像观察到的细胞内摄取(C) Cy5-siRNA 到 QD–PEI 和(C) Cy5-siRNA 到 QD–PEI–Hph-1。(D) QD–PEI 在体外培养过程中荧光共振能量传递（FRET）信号的强度。(E) 与 Cy5-siRNA 单独荧光强度相比，QD–PEI 在培养时间内的 FRET 强度。比例尺为 10μm。

Adapted with permission from ref. [86].
经过 86 号参考文献的许可进行了调整。

Other organic and inorganic nanoparticles
其他有机和无机纳米颗粒

Carbon nanotubes (CNTs), gold NPs, and silica NPs have inherently interesting properties that could be potential candidates for gene delivery and tracking. Although not common nanoparticles for gene delivery applications, these organic and inorganic NPs are becoming more popular for imaging and drug/gene delivery applications. Collectively, these particles exhibit simple surface functionalization, unique acoustic signals, and loadability.
碳纳米管（CNTs）、金 NPs 和二氧化硅 NPs 有固有的有趣特性，可能成为基因传递和追踪的潜在候选物。虽然不常用于基因递送应用的纳米颗粒，这些有机和无机 NPs 正在变得越来越受欢迎，用于成像和药物/基因递送应用。总的来说，这些颗粒具有简单的表面功能化、独特的声学信号和可装载性。

Carbon nanotubes 碳纳米管

Since the first publication of CNTs in 1991, these carbon nanomaterials have been widely explored for their many potential biomedical applications [95], [96], [97]. CNTs are one dimensional seamless cylindrical graphene sheets where single-walled carbon nanotubes (SWNTs) are composed of a single graphene sheet and multi-walled carbon nanotubes (MWNTs) are made up of multiple concentric SWNTs. Diameters of SWNTs can be as low as 0.4 nm while for MWNTs the diameter can be around 100 nm. Lengths typically range from hundreds of nanometers up to tens of micrometers. The unique feature of CNTs is the graphene wall that is easily functionalized with various biomolecules, imaging agents, and drugs. Gene delivery is made possible when these insoluble nanomaterials are either covalently functionalized by oxidation of the CNTs in acidic conditions and 1,3-dipolar cycloaddition reaction or noncovalently functionalized with hydrophobic or π–π stacking between the CNT and another non-polar ring such as the backbone of DNA. Ammonium-functionalized CNTs can bind plasmid DNA by electrostatic interactions and penetrate the cell membrane through a nanoneedle model as visualized by TEM [98]. In this way, CNTs are seen as exceptional nanomaterials for gene and drug delivery because they can be taken up by mammalian cells in an endocytosis-independent pathway. Both SWNTs and MWNTs have also been found to form stable complexes with plasmid DNA and allow for the successful delivery of genes [99], [100]. Due to the versatility of the CNT wall, fluorescent markers and biomolecules can be bound to study the cellular uptake efficiency. In this way, CNTs can be covalently linked with fluorescein or biotin to form a fluorescent biotin–avidin complex to study in vitro uptake [101].
自 1991 年首次发布 CNT 以来，这些碳纳米材料已被广泛用于许多潜在的生物医学应用[95]，[96]，[97]。CNT 是一维无缝圆柱状石墨烯片，其中单壁碳纳米管(SWNTs)由单层石墨烯片组成，多壁碳纳米管(MWNTs)由多个同心排列的 SWNTs 组成。SWNTs 的直径可以达到 0.4 纳米，而 MWNTs 的直径可达到约 100 纳米。长度通常范围从数百纳米到数十微米。CNT 的独特特征是石墨烯壁，可以轻松与各种生物分子、成像剂和药物功能化。当这些不溶性纳米材料通过酸性条件下 CNT 的氧化和 1,3-偶极环加成反应或与疏水性或π-π堆积之间的非共价功能化时，基因传递变为可能，比如 CNT 与 DNA 骨架之类的非极性环之间的π-π堆积。氨基功能化的 CNT 可以通过静电相互作用与质粒 DNA 结合，并通过在 TEM 下可视化的纳米针模型穿透细胞膜。这样一来，CNT 被视为卓越的基因和药物传递纳米材料，因为它们可以通过非内吞途径被哺乳动物细胞摄取。发现 SWNTs 和 MWNTs 也能与质粒 DNA 形成稳定的复合物，并成功地传递基因。由于 CNT 壁的多功能性，荧光标记物和生物分子可以结合以研究细胞摄取效率。这样一来，CNT 可以与荧光素或生物素共价结合，形成荧光生物素-亚维丁复合物以进行体外摄取研究。

As in vitro studies of DNA delivery have demonstrated successful cellular uptake and versatile functionalization of CNTs, siRNA bound to CNTs can be used for gene silencing. CNT-PEG-siRNA have been synthesized with efficient uptake and inhibition of the gene coding for lamin A/C protein in HeLa cells [102]. The knockdown efficiency was dependent on the siRNA linkage to CNT and a disulfide bond linkage allowed for the gene knockdown levels to reach about 70%. In vivo drug delivery of the anticancer drug such as paclitaxel and doxorubicin has been exemplified by PEGylated SWNTs with specific tumor targeting and minimal toxicity [103], but very few examples of in vivo gene delivery has been reported using CNTs [104], [105], [106], [107], [108]. Notably, pristine SWNTs were noncovalently bound to siRNA, which served as the delivery agent to silence hypoxia-inducible factor 1 alpha (HIF-1α) as well as the dispersing agent for SWNTs [104]. The complex was intratumorally administered into mice bearing pancreatic cells with a HIF-1α/luciferase reporter. Using this model, bioluminescence was used to image luciferase reporter gene activity in vivo and SWNTs demonstrated a high photon flux indicating the activity of HIF-1α. Therefore, if the activity of HIF-1α decreased then the luciferin bioluminescence signal had decreased intensity. As seen in Fig. 5, the bioluminescence signal after luciferin is injected to the tumor is high but with the addition of SWNTs complexed to HIF-1α siRNA, the bioluminescence signal of the mouse tumor decreases corresponding to significant HIF-1α inhibition. Recent works have shown the effective gene delivery of insulin 2 gene to mice using another carbon nanomaterial – fullerene, namely by tetra(piperazino)fullerene epoxide [109]. Individual SWNTs exhibit unique intrinsic properties such as NIR photoluminescence, strong Raman scattering, and photoacoustic signals that could be used to track gene delivery. Future works could enhance the interesting properties of CNTs for efficient gene delivery tracking.
体外研究表明，碳纳米管（CNTs）成功进行了 DNA 传递，可以对 CNTs 进行多功能化，使 CNTs 与 siRNA 结合进行基因沉默。CNT-PEG-siRNA 已被合成，并在 HeLa 细胞中有效摄取并抑制编码 lamin A/C 蛋白的基因。沉默效率取决于 siRNA 与 CNT 的连接，二硫键连接使基因沉默水平达到约 70%。使用 PEG 化 SWNTs 作为载体进行抗癌药物如紫杉醇和阿霉素的体内递送已被证明具有特异性肿瘤靶向和最小毒性，但使用 CNT 进行体内基因递送的案例很少。 SWNTs 与 siRNA 非共价结合，可用作抑制缺氧诱导因子 1α（HIF-1α）的递送剂以及 SWNTs 的分散剂。将该复合物肿瘤内注射到携带胰腺细胞的小鼠中，这些细胞具有 HIF-1α/荧光素酶报告基因。利用生物发光成像小鼠体内的荧光素酶报告基因活性，并且 SWNTs 显示出高的光子通量，表明 HIF-1α的活性。因此，如果 HIF-1α的活性降低，那么琼脂糖荧光强度会降低。图 5 显示，在将琼脂糖注射到肿瘤后生物发光信号较高，但加入与 HIF-1α siRNA 复合的 SWNTs 后，小鼠肿瘤的生物发光信号降低，对应于显著的 HIF-1α抑制。最近的研究表明，使用另一种碳纳米材料富勒烯对小鼠进行胰岛素 2 基因的有效基因递送。 单个单壁碳纳米管表现出独特的内在特性，如近红外光致发光、强拉曼散射和光声信号，可用于追踪基因传递。未来的研究可能会提高碳纳米管的有趣特性，以实现有效的基因传递追踪。

1. Download : Download high-res image (266KB)
   下载：下载高清图片（266KB）
2. Download : Download full-size image
   下载：下载全尺寸图像

Figure 5. (A) Schematic diagram of SWNT complexed with non-targeted siRNA (SWNT/siSC). (B) Tumor bearing mice were imaged 5 min after the addition of luciferin. Then the tumors were injected with siRNA targeting HIF-1 alpha (siHIF-1a), SWNT/siSC, and SWNT complexed with targeted siRNA for HIF-1 alpha (SWNT/siHIP-1a). (C) Photon flux was imaged during the treatments illustrating the HIF-1 inhibition and therefore the delivery effectiveness of siRNA.
图 5。(A) 单壁碳纳米管与非靶向 siRNA 复合物的示意图（SWNT/siSC）。（B）携带肿瘤的小鼠在加入琼脂素后的 5 分钟内成像。然后将肿瘤注射 siRNA 靶向 HIF-1 alpha（siHIF-1a）、SWNT/siSC 和单壁碳纳米管与靶向 HIF-1 alpha 的 siRNA 复合物（SWNT/siHIP-1a）。（C）成像记录治疗期间的光子通量，说明 HIF-1 抑制及 siRNA 传递效果。

Adapted with permission from ref. [104].
得到的许可编号[104]适配。

Gold nanoparticles 金纳米粒子

The rapid advancement of nanotechnology over the past decade has opened opportunities for the design of functional gold nanoparticles (AuNPs) for photothermal therapy, biosensing, molecular imaging, and gene therapy. Recently, AuNPs have been considered as excellent gene delivery systems due to their unique properties and excellent abilities to bind/bioconjugate biological ligands, DNA and siRNA through surface bonding [116], [117], [118], [119], [120]. Lee et al. reported biologically functional cationic phospholipid-gold NPs that simultaneously exhibit carrier capabilities, demonstrate improved colloidal stability, maintain plasmonic properties, and show good biocompatibility under physiological conditions [121].
过去十年内纳米技术的快速发展为设计功能性金纳米颗粒(AuNPs)提供了机会，用于光热治疗、生物传感、分子成像和基因治疗。最近，由于其独特的性质和优异的能力，AuNPs 被认为是优秀的基因传递系统，可以通过表面结合与生物配体、DNA 和 siRNA 结合/生物共轭[116]，[117]，[118]，[119]，[120]。Lee 等报道了具有生物功能的阳离子磷脂化金纳米粒子，同时具有载体能力，表现出改善的胶体稳定性，在生理条件下保持等离子性能，并表现出良好的生物相容性[121]。

AuNPs exhibit unique optical properties due to strong and tunable surface plasmon absorption in the NIR range, which can cause photothermal effects to trigger a variety of biological activities, such as NIR laser-induced release of therapeutic genes from the AuNPs. Chen et al. reported the remote control of gene expression in HeLa cells using AuNPs excited with NIR irradiation [21]. The GFP reporter gene was attached to the surface of AuNPs by linking of thiolated DNA through Au–S bonds. In addition, AuNPs were further labeled with Streptavidin-Alexa Fluor 647 to image AuNPs in living cells. When femtosecond NIR irradiation was applied to the AuNP–DNA conjugates, the shape of AuNPs was changed, and the gene was expressed. The transformation of shape might be induced by a release of DNA from AuNPs, as similar phenomenon was described by the Niidome group [122], [123]. Wijaya et al. also showed the use of gold nanorods to selectively release multiple DNA oligonucleotides [124]. Therefore, a controlled release system for gene delivery based on AuNPs offers great potency in gene therapy. Recently, Lu et al. reported NIR light-inducible NF-κB down regulation through folate receptor-targeted hollow gold nanospheres carrying siRNA recognizing NF-κB p65 subunit (Fig. 6) [125]. The targeted AuNPs exhibited significantly high tumor uptake in a mouse model of cervical cancer as imaging by micro-PET/computed tomography. Controlled cytoplasmic delivery of siRNA was possible by the AuNP photothermal effect after NIR light irradiation. Efficient down regulation of NF-κB p65 was achieved only in tumors irradiated with NIR light. Heat or cavitation-induced endosomal membrane disruption and siRNA diffusion into the cytosol can be monitored by Cy3 fluorescence.
金纳米粒子(AuNPs)由于在 NIR 范围内具有强大和可调谐的表面等离子吸收而表现出独特的光学性质，这可以引发光热效应，从而触发各种生物活动，例如通过 NIR 激光诱导 AuNPs 释放治疗基因。陈等报道了使用受激 NIR 辐照的 AuNPs 在 HeLa 细胞中远程控制基因表达的研究。绿色荧光蛋白报告基因通过巯基化 DNA 与 Au–S 键连接到 AuNPs 表面。此外，AuNPs 还进一步标记了 Streptavidin-Alexa Fluor 647 以在活细胞中成像 AuNPs。当飞秒 NIR 辐照被应用到 AuNP-DNA 共价物上时，AuNPs 的形状发生了变化，基因被表达。形状的改变可能是由于 DNA 从 AuNPs 释放引起的，与 Niidome 等描述的现象类似。Wijaya 等还展示了使用金纳米棒选择性释放多个 DNA 寡核苷酸。因此，基于 AuNPs 的基因运输的受控释放系统在基因治疗中具有巨大的潜力。最近，卢等报道了通过叶酸受体靶向的载有 siRNA 识别 NF-κB p65 亚基的中空金纳米球的 NIR 光诱导 NF-κB 下调。靶向 AuNPs 在宫颈癌小鼠模型中表现出明显高的肿瘤摄取，在显微 PET/计算机断层扫描中成像。通过 NIR 光照射后，AuNP 光热效应使 siRNA 的胞质传递得以控制。只有受 NIR 光照射的肿瘤中才能有效下调 NF-κB p65 的表达。通过 Cy3 荧光可以监测热或空化引起的溶骨体膜破裂和 siRNA 扩散至细胞质。

1. Download : Download high-res image (714KB)
   下载：下载高清图像（714KB）
2. Download : Download full-size image
   下载：下载全尺寸图像

Figure 6. Scheme for bioconjugation of HAuNS-siRNA and photothermal-induced siRNA release. (A) Scheme for the synthesis of F-PEG-HAuNS-siRNA and their proposed intracellular itinerary following NIR light irradiation. (B) Photothermal-induced endolysosomal escape of Dy547-labeled siRNA.
图 6. HAuNS-siRNA 的生物共轭化方案及光热诱导 siRNA 释放方案。(A) F-PEG-HAuNS-siRNA 合成方案及其在 NIR 光照射后的胞内可能路径示意图。(B) 光热诱导的 Dy547 标记的 siRNA 内溶酶体逃逸。

Adapted with permission from ref. [125].
根据参考文献[125]获得授权后进行了修改。

Braun et al. reported a novel lipid-based gold NP that provides temporally and spatially controlled cellular delivery of siRNA for gene silencing, through a direct endosomal release mechanism activated by pulsed laser treatment (Fig. 7) [126]. The NIR laser was able to release Cy3-labeled siRNA, which was conjugated to the surface of gold NPs. Fluorophores in close proximity to gold NP can be efficiently quenched by nanoparticle-based surface energy transfer (NSET) mechanism [127]. The Cy3 dye is partially quenched when near the gold, laser-mediated release and delivery of siRNA can be monitored after dequenching of Cy3. In addition, a Tat peptide-lipid coating allows the use of low NP concentrations and laser-dependent endosomal RNA release. The photothermal transfection technique may be used in the rational design of targeted gene delivery systems for successful gene therapy and imaging.
Braun 等人报道了一种新型基于脂质的金纳米颗粒，通过脉冲激光处理激活的直接内体释放机制实现了 siRNA 在细胞内的时间和空间受控输送，用于基因沉默（图 7）[126]。红外激光能够释放与金纳米颗粒表面结合的 Cy3 标记的 siRNA。金纳米颗粒附近的荧光物质可以通过基于纳米颗粒表面能量转移（NSET）机制有效猝灭[127]。当 Cy3 染料靠近金时，部分猝灭，激光介导释放和传递 siRNA 可在 Cy3 变为解猝后进行监测。此外，Tat 肽脂质包衣使得可以使用低浓度的纳米颗粒和激光依赖的内体 RNA 释放。光热转染技术可以用于有针对性的基因传递系统的合理设计，从而实现成功的基因治疗和成像。

1. Download : Download high-res image (577KB)
   下载：下载高清晰度图片（577KB）
2. Download : Download full-size image
   下载：下载全尺寸图像

Figure 7. (A) Diagram of Tat-lipid-coated siRNA used for transfection and selective release of siRNA and (B) schematic of the siRNA construct.
图 7。 (A) Tat-脂质包被的 siRNA 示意图（用于转染和选择性释放 siRNA），(B) siRNA 构型原理图。

Adapted with permission from ref. [126].
经过许可后修改自参考文献 [126]。

Advancements in nanotechnology continue to introduce inorganic and organic nanomaterials for molecular imaging and gene delivery. Here AuNPs, silica NPs, and CNTs have been introduced as future gene delivery materials. The unique intrinsic properties of these nanomaterials are utilized to image gene delivery and future works can combine such materials for synergistic effects.
纳米技术的进展不断推出无机和有机纳米材料用于分子成像和基因传递。其中金纳米颗粒（AuNPs）、二氧化硅纳米颗粒和碳纳米管被用作未来的基因传递材料。这些纳米材料的独特内在性能被利用于成像基因传递，未来的工作可以结合这些材料以产生协同效应。

Conclusions and future perspectives
结论和未来展望

NPs are particularly useful for molecular imaging of gene delivery due to their unique physicochemical properties. With advances in nanotechnology, functional NPs deserve significant research efforts as they can be integrated for quantitative, noninvasive imaging and targeted gene therapy within one entity. The ultimate goal is that functional NPs allow for efficient, specific in vivo delivery of genes without systemic toxicity, and the dose delivered as well as the therapeutic efficacy can be accurately measured noninvasively and spatiotemporally.
由于纳米粒子独特的物理化学性质，它们特别适用于基因传递的分子成像。随着纳米技术的进步，功能性纳米粒子值得进行重要的研究工作，因为它们可以被整合用于定量、非侵入性成像和靶向基因治疗。最终目标是，功能性纳米粒子能够实现基因的有效、特异性体内传递，而无系统毒性，并且可以准确测量非侵入性和时空特性下的给药剂量以及治疗效果。

However, looking into the future, it is imperative to have a better understanding of the basic principles involved in designing and applying NPs for diagnosis, treatment, or the combination of imaging and therapy in different clinical situations. Many factors need to be optimized to design advanced NPs for molecular imaging of gene delivery, among which are biocompatibility, pharmacokinetics, in vivo targeting efficacy, and cost-effectiveness. Foremost, minimizing the potential toxicity of NPs is critically important. In order to validate the potential of therapeutic gene delivery with NPs, in vitro cytotoxicity and nanoparticle effects have been examined to a large extent. Generally, the toxicity has a strong dependency on the physicochemical properties of NPs, such as size, surface charge, and surface coating materials, in addition to the dosage of NPs and the duration of exposure. However, with regards to potential cytotoxic effects of NPs, many apparently contradicting results have been obtained due to the different cell types/animal models tested, the variety in type of materials utilized, and the variability in concentrations of the NPs used. The modifications to reduce cytotoxicity may also compromise the functionality of the NPs. For example, modification of the NPs with PEG may improve aqueous dispersion, prevent aggregation, but it will also significantly reduce cellular uptake [93].
然而，展望未来，必须更好地理解设计和应用纳米颗粒用于不同临床情况中的诊断、治疗或影像与治疗结合的基本原理至关重要。需要优化许多因素来设计用于基因传递分子成像的先进纳米颗粒，其中包括生物相容性、药代动力学、体内靶向效果和成本效益。首要的是最大限度地减少纳米颗粒的潜在毒性。为了验证用于纳米颗粒的治疗基因传递的潜力，已经在很大程度上检查了体外细胞毒性和纳米颗粒效应。通常，毒性与纳米颗粒的物理化学特性有很大关联，比如大小、表面电荷和表面涂层材料，除了纳米颗粒的剂量和暴露时长。然而，关于纳米颗粒的潜在细胞毒性效应，由于测试的不同细胞类型/动物模型、使用的材料类型的多样性以及使用的纳米颗粒浓度的可变性，获得了许多看似矛盾的结果。为减少细胞毒性而进行的修改也可能损害纳米颗粒的功能。例如，使用 PEG 改性纳米颗粒可能会改善水分散性，防止聚集，但也会显著降低细胞摄取量。

Second, the specificity of NPs for selected cells and tissues is critical in both diagnostic imaging and gene therapy. Antibodies, peptides, aptamers and small organic molecules, have been used in NP systems as targeting agents for specific biomarkers on target cells. The development of novel noninvasive imaging in combination with biomarker targeted NPs has the potential for early detection of diseases and determination of the responses to gene therapy. However, linking biomarkers with disease behavior and personalized treatment remain a significant challenge due to the heterogeneous nature of most diseases. While targeting agents enable NP binding affinity/specificity, the type of ligand and method of NP attachment can significantly affect its targeting ability [128]. For instance, a functional group modification of the NPs during conjugation may change their chemical properties by embedding part of the ligand binding site in NPs therefore decreasing the targeting capabilities. Knowledge of how to tune and exactly control the ratio between imaging labels and targeting units on functional nanomaterials can lead to improved system design and predictions of structure, function, and activity of the generated NPs [129], [130]. In addition to ligand bioactivity, these molecules also may affect NP stability and immunogenicity. For example, antibodies/proteins derived from non-human animal sources can create the possibility of unwanted immune responses.
NPs 对于选择性细胞和组织的特异性对于诊断成像和基因治疗至关重要。抗体、肽、寡核苷酸和小有机分子等在 NP 系统中被用作靶向代理，与目标细胞上的特定生物标志物配对。将新型非侵入性成像技术与生物标志物靶向 NP 相结合，具有早期疾病检测和基因治疗反应的潜力。但由于大多数疾病的异质性，将生物标志物与疾病行为和个性化治疗联系起来仍然是一个重要挑战。靶向代理可以提高 NP 结合亲和力和特异性，配体的类型和 NP 附着方法也会显著影响其靶向能力。例如，在共轭过程中对 NP 进行功能基团修饰可能改变其化学性质，通过将部分配体结合位点嵌入 NP 中，从而降低了靶向能力。了解如何调节和准确控制功能纳米材料上成像标记和靶向单元比例可以改进系统设计，并对生成的 NP 的结构、功能和活性进行预测。除了配体生物活性，这些分子也可能影响 NP 的稳定性和免疫原性。例如，来自非人类动物源的抗体/蛋白可能导致意外的免疫反应。

Third, the generation of multifunctional NPs for multimodality imaging will extend the limits of current molecular diagnostics and permit accurate diagnosis as well as the development of targeted gene delivery. A single probe helps to ensure the same pharmacokinetics and colocalization of signal for each modality and it also can avoid putting the additional stress on the body's blood clearance mechanisms that can accompany administration of multiple doses of probes [18], [131]. However, future work will have to address the issue of sensitivity and determine the detection thresholds for the different modalities. For example, PET is a highly sensitive imaging modality that requires the introduction of only a trace amount of probes, whereas a relatively high amount of probes needed for MRI in current systems limits the unique sensitivity of the PET. Thus, the detection sensitivities for different imaging modalities should be further considered and optimized. Additionally, the biodistribution of therapeutics exhibits changes on time scales of seconds to minutes. To ensure that a subject is being imaged in the same physiologic state and to correlate changes over time in the different modalities’ signals in response to an intervention, data must be acquired simultaneously or at least in very rapid succession. In designing an integrated scanner for simultaneous imaging, an obvious challenge relates in which the different modalities can interfere with each other, leading to major artifacts and/or image degradation.
第三，为多模态成像生成多功能纳米颗粒将扩展目前分子诊断的限制，实现准确诊断和靶向基因传递的发展。单一探针有助于确保每种模式的药代动力学和信号共定位，并且还可以避免给体内血液清除机制带来额外压力，后者可能伴随多剂量探针的给药[18] [131]。不过，未来的工作将需要解决敏感性问题，并确定不同模式的检测阈值。例如，PET 是一种高灵敏度成像模式，只需引入微量探针，而 MRI 在目前系统中所需的探针量相对较高，限制了 PET 的独特灵敏度。因此，应进一步考虑和优化不同成像模式的检测灵敏度。此外，治疗药物的生物分布在秒到分钟的时间尺度上发生变化。为了确保对被检者进行同时成像，并且在不同模式信号随时间变化以响应干预的情况下进行相关性分析，数据必须同时获取，或至少在非常快速的时间内获取。在设计用于同时成像的综合扫描仪时，一个明显的挑战是不同模式之间可能会相互干扰，导致严重伪影和/或图像退化。

Finally, gene therapy imaging to monitor therapeutic effects requires monitoring the changes of transduced/transfected target cells in terms of their location and number over time [132]. Unfortunately, till now, imaging techniques are typically used to visualize the delivery of fluorescently labeled siRNAs or carriers at the targeted site. However, it is not clear how a therapeutic response can be effectively monitored, as labeled siRNA or nanoparticles will fluoresce inside or outside target cells without transfection and gene expression. Therefore, more sophisticated imaging techniques need to be developed to monitor therapeutic responses induced by efficiently delivered therapeutic genes. In this point of view, utilizing reporter gene-based imaging techniques in combination with nanoplatforms introduced in this article can be an alternative choice. The expression of a transferred gene in vivo can be visualized indirectly by using imaging reporter gene, especially optical reporter genes. For the evaluation and optimization of gene therapy based on reporter gene imaging, the development of a novel gene carrier with controllable gene expression system coupled to a imaging reporter gene that can be monitored noninvasively and simultaneously is critical [133], [134]. Ideally, plasmid DNA should first be compacted into functional nanoparticles to form stable formula for targeted gene delivery and capable of providing protection against enzymatic, and thereby, the development of gene carrier may provide improved interpretation of therapeutic responses and thus allow optimization of novel gene therapeutic strategies.
基因治疗成像技术的最终目标是监测治疗效果，需要随着时间的推移监测转导的靶细胞的位置和数量变化。然而，目前的成像技术通常用于可视化在靶向位点传递的荧光标记 siRNA 或载体的过程。然而，目前尚不清楚如何有效监测治疗反应，因为标记的 siRNA 或纳米颗粒会在转染和基因表达的情况下在靶细胞内部或外部发出荧光。因此，需要开发更复杂的成像技术来监测通过有效传递治疗基因引起的治疗反应。在这个角度而言，利用基于报告基因成像技术结合本文介绍的纳米平台可能是一种替代选择。通过使用成像报告基因，尤其是光学报告基因，可以间接可视化体内转移基因的表达。对基于报告基因成像的基因治疗进行评估和优化，开发一个新型基因载体，具有可控的基因表达系统，配备一个成像报告基因，实现无创同时监测是至关重要的。理想情况下，质粒 DNA 应首先被压缩成功能性纳米颗粒，形成稳定的配方，用于靶向基因传递，并能够提供对酶的保护，因此，基因载体的发展可能提供对治疗反应的改进解释，从而实现新型基因治疗策略的优化。

Over a decade ago Dr. Verna said, “There are only three problems in gene therapy, delivery, delivery and delivery” [135]. Now in the new millennium, as gene therapy moves beyond viral vectors and into the nanotechnology field, there are at least three additional problems – toxicity, good manufacturing practice (GMP), and regulatory affairs. To date, the United States Food and Drug Administration (FDA) has not approved human gene therapy for clinical use. However, as nanoparticle gene therapy research continues to develop with standardized nanomaterial characterization and clinical trial efficacy, FDA is actively involved in overseeing the fast growth of such research. Being the sole regulatory affair administration for human drug approval, the FDA has partnered with numerous agencies to keep up with gene delivery and nanotechnology research. One such partnership is to include the National Institutes of Health (NIH), to launch the Genetic Modification Clinical Research Information System, providing transparent information of all ongoing clinical research for gene delivery. Other Public–Private Partnerships have also been set up to promote the understanding of the biological interactions of nanoscale materials. One critical partnership include FDA–National Cancer Institute (NCI)–National Institute of Standards and Technology (NIST) to establish the Nanotechnology Characterization Laboratory to speed the development of effective medical products with nanoengineered products. However, the regulatory hurdles to gene therapy, such as safety and efficacy, remain roadblocks in the clinical application of gene therapy. The future of functional NP design for molecular imaging and gene delivery mainly depends on multidisciplinary cooperation between molecular biologists, chemists, physicists, materials scientists, and imaging specialists. With continuous efforts by multidisciplinary approaches, the use of such nanoplatforms will shed new light on molecular diagnostics, gene therapy, and personalized medicine.
十多年前，Verna 博士说过：“基因疗法只有三个问题，传递、传递和传递”。现在新千年到来之际，随着基因疗法超越病毒载体进入纳米技术领域，至少又出现了三个新问题——毒性、良好的生产规范（GMP）和监管事务。截至目前，美国食品药品监督管理局（FDA）尚未批准人类基因疗法用于临床应用。然而，随着纳米颗粒基因疗法研究的发展，通过规范化纳米材料表征和临床试验效果，FDA 正积极参与监督这一研究的快速增长。作为唯一的人类药物批准监管机构，FDA 已与许多机构合作，以跟进基因传递和纳米技术研究。其中之一是与国家卫生研究院（NIH）合作，共同推出基因修饰临床研究信息系统，提供所有进行中基因传递临床研究的透明信息。还设立了其他公私合作伙伴关系，促进纳米尺度材料的生物相互作用的理解。一个重要的合作伙伴关系包括 FDA-国家癌症研究所（NCI）-国家标准技术研究所（NIST），建立纳米技术表征实验室，推动具有纳米工程产品的有效医疗产品的发展。然而，基因疗法的监管障碍，例如安全性和有效性，仍然是基因疗法的临床应用中的障碍。 分子成像和基因传递功能性纳米粒子设计的未来主要取决于分子生物学家、化学家、物理学家、材料科学家和成像专家之间的跨学科协作。通过跨学科方法的持续努力，这些纳米平台的使用将为分子诊断、基因治疗和个性化医学带来新的启示。