Even though there are alternatives and similar platforms, such as yeast display or ribosome display technology, phage display is still considered the best option due to its high diversity and easy handling [210,211,212,213,214].
尽管还有其他类似的平台，如酵母展示或核糖体展示技术，但噬菌体展示因其多样性高和易于操作，仍被认为是最佳选择[ 210, 211, 212, 213, 214]。
As stated earlier, the BBB prevents roughly 98% of potential drugs, pathogens, and molecules from entering the CNS. However, bacteriophages modified with the phage display technique, presenting specific receptors on the surface of their capsids, can penetrate the BBB using the Trojan horse mechanism [215,216]. Moreover, the endocytosis-based mechanism was also confirmed for bacteriophages (Figure 2B) [13,123,217]. The E.coli bacteriophage PK1A2 was demonstrated to bind to polysialic acid on the surface of human neuroblastoma cells, thus sharing structural similarity with the E.coli phage receptor [218,219]. Not only did the phages bind to eucaryotic cells, but they were also internalized into the cells through endocytosis and largely retained infectivity for 24 h [219]. Anand et al. developed a bifunctional viral nanocontainer based on the Salmonella-specific bacteriophage P22 [31]. Ziconotide, an analgesic peptide drug derived from a marine snail, was incorporated into the nanocontainer, while a cell-penetrating peptide HIV-tat was displayed on the exterior surface of the phage capsid. Such a modified P22 was successfully transferred into several cell-based models of rat brain microvascular endothelial cells as well as human brain microvascular endothelial cells. Hence, these findings open another alternative route for drug administration [31]. The F88 phage was also reported to easily penetrate the BBB after intranasal administration. One of the most advantageous features of filamentous bacteriophages is their shape and structure. The linear structure of f88 makes it a favorable nanocarrier to penetrate the BBB. After the intranasal administration of f88, phage particles were detected in the olfactory bulb and hippocampus regions of mice brains [220].
如前所述，BBB 阻止了大约 98% 的潜在药物、病原体和分子进入中枢神经系统。然而，利用噬菌体展示技术改造的噬菌体在其外壳表面呈现出特异性受体，可以利用特洛伊木马机制穿透 BBB [ 215, 216]。此外，噬菌体的内吞机制也得到了证实（图 2B）[13, 123, 217]。大肠杆菌噬菌体 PK1A2 被证明能与人类神经母细胞瘤细胞表面的多聚硅酸结合，因此与大肠杆菌噬菌体受体具有结构相似性[218，219]。噬菌体不仅能与真核细胞结合，还能通过内吞作用内化到细胞中，并在 24 小时内基本保持感染性[ 219]。Anand 等人开发了一种基于沙门氏菌特异性噬菌体 P22 的双功能病毒纳米容器[ 31]。在纳米容器中加入了从海洋蜗牛中提取的镇痛多肽药物 Ziconotide，同时在噬菌体外壳表面显示了细胞穿透肽 HIV-tat。这种经过修饰的 P22 成功地转移到了几种基于细胞模型的大鼠脑微血管内皮细胞和人类脑微血管内皮细胞中。因此，这些发现开辟了另一种给药途径[ 31]。另据报道，F88噬菌体经鼻内给药后可轻易穿透生物BB。丝状噬菌体最有利的特征之一是其形状和结构。F88 的线性结构使其成为穿透 BBB 的有利纳米载体。鼻内给药 f88 后，在小鼠大脑的嗅球和海马区检测到了噬菌体颗粒[ 220]。
A novel approach using a convection-enhanced delivery method (CED) was used by Ksendzovsky et al., in which a high-pressure gradient of bulk flow is generated as a delivery tool for a desired preparation [221]. The tip of an infusion catheter delivers therapeutics directly through the interstitial spaces of the CNS. By applying this, we eliminate the requirement to deliver high, unwieldy therapeutic concentrations, which enables us to achieve therapeutic concentration of the drug in the brain parenchyma [222]. The results from Ksendzovsky et al.’s experiments indicate that CED is a promising method of phage delivery to the brain due to the fact that the M13 bacteriophage was spotted in the white and grey matter of mice brains.
Ksendzovsky 等人采用了对流增强给药法（CED）这一新颖方法，在这种方法中，高压梯度的大量流动被用作所需制剂的给药工具[221]。输液导管的尖端直接通过中枢神经系统的间隙输送治疗药物。通过应用这种方法，我们不再需要输送高浓度的治疗药物，从而能够在脑实质内达到治疗浓度[222]。Ksendzovsky 等人的实验结果表明，由于在小鼠大脑的白质和灰质中发现了 M13 噬菌体，CED 是一种很有前景的向大脑输送噬菌体的方法。
The development of CNS-related diseases is much more challenging than that of non-CNS diseases due to the presence of the BBB, but also the brain–blood tumor barrier (BBTB). Although the structure itself differs from the BBB, both the BBB and BBTB drastically limit the efficient transport of most currently available drugs, mainly due to their molecular size, which surpasses the threshold limiting the molecules that can readily penetrate BBTS and BBB [223,224]. Additional challenges in CNS drug development include the high costs associated with drugs capable of penetrating the BBB and a potentially increased risk of side effects. Compared to other drugs, those targeting the CNS require higher concentrations due to the indirect route to the CNS via the liver and kidneys. These organs tend to remove a significant portion of these molecules from the bloodstream. One approach to improve drug delivery is through receptor-mediated transcytosis, a method that targets drugs to specific receptors expressed in the BBB. To date, two such monoclonal antibody-based constructs—with either a transferrin receptor or insulin receptor—have successfully completed phase II clinical trials [225,226].
由于存在脑血管屏障（BBB）和脑血肿瘤屏障（BBTB），中枢神经系统相关疾病的发展比非中枢神经系统疾病更具挑战性。虽然脑血肿瘤屏障本身的结构与脑血屏障不同，但脑血肿瘤屏障和脑血肿瘤屏障极大地限制了大多数现有药物的有效转运，主要原因是药物的分子大小超过了能轻易穿透脑血肿瘤屏障和脑血肿瘤屏障的分子阈值[ 223, 224]。中枢神经系统药物开发面临的其他挑战包括：能够穿透 BBB 的药物成本高昂，副作用风险可能增加。与其他药物相比，以中枢神经系统为靶点的药物需要更高的浓度，这是因为药物需要通过肝脏和肾脏间接进入中枢神经系统。这些器官往往会从血液中清除很大一部分这些分子。改善给药的一种方法是通过受体介导的转囊作用，这种方法将药物靶向表达在 BBB 中的特定受体。迄今为止，两种基于单克隆抗体的构建物–转铁蛋白受体或胰岛素受体–已经成功完成了 II 期临床试验[ 225, 226]。
During neurological disorders, the expression of different unique receptors that home in on the CNS can be used to improve drug delivery, and such peptides can be selected with phage display technology [227]. To list a few examples, Eriste et al. identified a novel peptide gHo that effectively binds glioma cells [228] and, when covalently conjugated to the cell-penetrating peptide pVEC and doxorubicin, showed promise in the mouse subcutaneous U87 tumor model, although no effect was demonstrated in the intracranial glioma model [228,229,230]. Chen et al. applied phage display to identify an M1 peptide that localized to the brain and was demonstrated to penetrate the BBB/BBTB [231]. Furthermore, conjugation of paclitaxel to the RGD-modified M1 peptide not only allowed for efficient translocation across the BBB, but also greatly improved—typically low—PTX solubility. Importantly, it was the first vector targeting glioma with the ability to travel through the BBB and BBTS. Kim et al. used in vitro and in vivo phage display screens to select glioblastoma targeting peptides and identified a Cadherin 2-binding peptide with the ability to selectively target GSCs over differentiated glioma and non-neoplastic brain cells [232]. Moreover, in a mouse model of intracranially xenografted glioblastoma, the peptide—administered intravenously—specifically targeted intracranial tumors. Phage display technology has also been used to develop nanobodies targeting CA9 [233] and ABCC3 [234], expression of which was detected to correspond with the worse overall survival of patients with glioblastoma. Another approach could involve combining the use of phage display for identification of tumor-targeting peptides and CAR-T therapy for novel modifications of classic CAR-T, which seems to have potential in the development of novel cytotoxic therapies for glioblastoma, as demonstrated by Potez et al. [202].
在神经系统疾病中，中枢神经系统内不同独特受体的表达可用于改善给药效果，此类多肽可通过噬菌体展示技术筛选出来[ 227]。举几个例子，Eriste 等人发现了一种新型多肽 gHo，它能有效结合胶质瘤细胞[228]，与细胞穿透肽 pVEC 和多柔比星共价结合后，在小鼠皮下 U87 肿瘤模型中显示出良好的效果，但在颅内胶质瘤模型中没有显示出效果[228, 229, 230]。Chen 等人应用噬菌体展示技术鉴定出一种 M1 肽，这种肽能定位到大脑，并被证明能穿透 BBB/BBTB[231]。此外，将紫杉醇与 RGD 修饰的 M1 肽连接，不仅能有效转运穿过 BBB，还大大提高了通常较低的 PTX 溶解度。重要的是，这是首个能够穿越 BBB 和 BBTS 的胶质瘤靶向载体。Kim 等人利用体外和体内噬菌体展示筛选技术筛选出胶质母细胞瘤靶向肽，并鉴定出一种与 Cadherin 2 结合的肽，该肽能够选择性地靶向胶质细胞间充质干细胞，而不是分化的胶质瘤和非肿瘤性脑细胞[ 232]。此外，在小鼠颅内异种移植胶质母细胞瘤模型中，静脉注射该肽可特异性靶向颅内肿瘤。噬菌体展示技术还被用于开发靶向 CA9 [ 233] 和 ABCC3 [ 234] 的纳米抗体。另一种方法是结合使用噬菌体展示技术鉴定肿瘤靶向肽和CAR-T疗法对经典CAR-T进行新的改造，正如Potez等人[ 202]所证明的那样，这种方法在开发胶质母细胞瘤的新型细胞毒性疗法方面似乎具有潜力。
Importantly, the potential of phage display is not limited to being a useful platform in the drug discovery process—modified phage particles can also be directly applied as targeted delivery platforms, especially because natural phages generally lack native tropism to eukaryotic cells. Przystal et al. report constructing a dual targeting hybrid vector, containing the genome of human adeno-associated virus (AAV) incorporated into the engineered particles of bacteriophage M13 [32]. The vector was intended for intravenous administration, and its dual tumor targeting system relied on, firstly, targeting αvβ3 integrin receptor by phage-expressed ligand RGD4C, and secondly, tumor-specific and temozolomide-induced gene expression from a Grp78 promotor, acting as suicide gene therapy. The authors demonstrated the potential of this approach in vivo, as the combination of temozolomide and the hybrid vector suppressed the growth of orthotopic glioblastoma in mice.
重要的是，噬菌体展示的潜力不仅限于成为药物发现过程中的有用平台–经过修饰的噬菌体颗粒还可以直接用作靶向递送平台，特别是因为天然噬菌体通常缺乏对真核细胞的原生滋养性。Przystal 等人报告说，他们构建了一种双靶向混合载体，将人类腺相关病毒（AAV）的基因组整合到噬菌体 M13 的工程颗粒中[ 32]。该载体用于静脉注射，其双重肿瘤靶向系统首先依赖于靶向α v 噬菌体表达的配体 RGD4C 靶向 β 3 整合素受体；其次，肿瘤特异性和替莫唑胺诱导的基因表达来自 Grp78 启动子，起到自杀式基因治疗的作用。作者证明了这一方法在体内的潜力，因为替莫唑胺和混合载体的结合抑制了小鼠正位胶质母细胞瘤的生长。
Many aspects of phage interactions with the nervous system still remain unexplained, i.e., phage binding to the tumor cells, their delivery, and phage titer after its administration (pharmacokinetics). Moreover, it is yet unknown whether they are sufficient to treat tumors in human.
噬菌体与神经系统相互作用的许多方面仍未得到解释，例如噬菌体与肿瘤细胞的结合、噬菌体的输送以及给药后的噬菌体滴度（药代动力学）。此外，噬菌体是否足以治疗人类肿瘤也还是未知数。
While all these examples focus primarily on the development of novel anti-cancer therapies, it is important to note that the peptides identified with the phage display technology or modified phage particles can also be used for the development of new strategies for the diagnosis and imaging of tumor cells.
虽然所有这些例子都主要集中在新型抗癌疗法的开发上，但必须指出的是，利用噬菌体展示技术或改良噬菌体颗粒鉴定出的肽也可用于开发肿瘤细胞诊断和成像的新策略。