6. Intranasal Administration
6.鼻内给药
A different approach to the problem of limited penetration through the BBB was suggested by Wan et al., who focused on bypassing the BBB altogether by choosing a different administration route and simultaneously using a peptide-homing tag, which would theoretically allow issues with the migration of drug molecules to the CNS to be overcome [235]. Intranasal application was proposed as a possible efficient alternative, and the M13 phage display platform was used to screen a peptide library for promising carriers that could then be intranasally administered to rats. Clone7 was selected for further examination based on the highest translocation efficiency—it demonstrated an approximately 50-fold higher translocation efficiency compared to the control phage. The 11 amino acids long sequence of the peptide from Clone7 was identified to be as follows: ACTTPHAWLCG. When administered intranasally to Wistar rats, Clone7 was detectable in the brain in a dose-dependent manner after 15 min, reaching maximum after 45 min, and a higher phage titer was detected in the brain than in the liver, spleen, and blood of the examined rats, indicating that Clone7 was able to bypass the BBB and enter the brain directly. Most of the Clone7 phage particles, as well as the peptide itself, were found along the olfactory nerve. Two possible direct pathways may exist to transport administrated substances from olfactory mucosa into the CNS: the olfactory nerve pathway and the olfactory epithelial pathway (Figure 2A) [236]. The results described above indicate that phages displaying the homing peptide entered the CNS through the olfactory nerve pathway [235]. Interestingly, when administered alone and not on a phage particle, the selected peptide—although clearly observed in the olfactory nerve—could not be detected in the brain in the given time frame of up to 8 h. To date, it has only been observed in the case of a single peptide; however, if this was a more general characteristic of nose-to-brain homing peptides, it would further enforce the application of phage particles as nanocarriers for targeted drug delivery to the brain following intranasal administration. Therefore, this discovery opens a possible new route of drug administration in the treatment of bacterial and viral infections of the CNS and also supports the potential of phage-based solutions in the treatment of brain cancers.
Wan 等人提出了一种不同的方法来解决 BBB 穿透力有限的问题，他们的重点是通过选择不同的给药途径并同时使用肽归宿标签来完全绕过 BBB，这样理论上就能克服药物分子向中枢神经系统迁移的问题[ 235]。研究人员提出鼻内给药是一种可能的高效替代方法，并利用 M13 噬菌体展示平台筛选了一个肽库，寻找有前景的载体，然后对大鼠进行鼻内给药。克隆 7 的转运效率最高–与对照噬菌体相比，它的转运效率大约高出 50 倍，因此被选为进一步研究的对象。经鉴定，克隆 7 的肽的 11 个氨基酸长序列如下：ACTTPHAWLCG。给 Wistar 大鼠鼻内注射 Clone7 后，15 分钟后可在大脑中检测到噬菌体，检测结果呈剂量依赖性，45 分钟后达到最大值，且在大脑中检测到的噬菌体滴度高于肝脏、脾脏和血液中的滴度，这表明 Clone7 能够绕过 BBB 直接进入大脑。大多数克隆7噬菌体颗粒以及肽本身都是沿着嗅神经发现的。从嗅粘膜向中枢神经系统输送给药物质可能有两条直接途径：嗅神经途径和嗅上皮途径（图 2A）[ 236]。上述结果表明，含有归巢肽的噬菌体是通过嗅觉神经通路进入中枢神经系统的[ 235]。有趣的是，当单独给药而不是在噬菌体颗粒上给药时，虽然在嗅觉神经中能清楚地观察到所选的肽，但在给定的长达 8 小时的时间框架内，在大脑中却检测不到。因此，这一发现为治疗中枢神经系统的细菌和病毒感染开辟了一条新的给药途径，同时也证明了噬菌体解决方案在治疗脑癌方面的潜力。
There are also studies of phages with two large, different exogenous peptides that were successfully created in a so-called dual phage display system. The first one, alkaline phosphatase, or green fluorescent protein (GFP), was located on the head, and the other, anti-CEA scFv, on the tail of the lambda phage [237]. A similar approach was used by Rajaram et al., who also created a dual-display system [238] in which a biomarker and a short peptide containing the streptavidin-binding motif Histidine-Proline-Glutamine (HPQ) were displayed on the opposite ends of the M13 bacteriophage capsid. This additional second peptide with a high affinity to streptavidin was introduced to allow for sample (phage) purification on streptavidin-conjugated magnetic beads to increase assay sensitivity and decrease background signal. Finally, Fagète et al. reported the selection of bispecific antibodies, which they described as a new frontier in antibody therapy, that also used a dual-phage system, allowing for the co-selection of pairs of antibodies based on the co-engagement of their respective targets [239]. The authors demonstrated that the use of two complementary leucine zipper domains, which later heterodimerize with a high affinity, allowed for the assembly of two antibody fragments—one displayed on the surface of the phage and the other present in the bacterial cell as a soluble component.
还有一些关于噬菌体的研究，在所谓的双噬菌体展示系统中，成功地制造出了带有两种大型、不同外源肽的噬菌体。第一种是碱性磷酸酶或绿色荧光蛋白（GFP），位于λ噬菌体的头部，另一种是抗癌胚抗原 scFv，位于λ噬菌体的尾部[ 237]。Rajaram 等人也使用了类似的方法，他们还创建了一个双显示系统[ 238]，其中生物标记物和含有链霉亲和素结合基团组氨酸-脯氨酸-谷氨酰胺（HPQ）的短肽被显示在 M13 噬菌体外壳的两端。引入对链霉亲和素具有高亲和力的第二肽是为了在链霉亲和素结合的磁珠上对样品（噬菌体）进行纯化，以提高检测灵敏度并减少背景信号。最后，法盖特（Fagète）等人报道了双特异性抗体的选择，他们将其描述为抗体疗法的一个新领域，该疗法也使用了双噬菌体系统，允许根据各自靶标的共同参与来共同选择成对抗体[ 239]。作者证明，使用两个互补的亮氨酸拉链结构域（它们随后会以高亲和力异源二聚化）可以组装出两个抗体片段–一个显示在噬菌体表面，另一个作为可溶性成分存在于细菌细胞中。
Carrera et al. (2004) prepared phage displaying scFv GNC 92H2, which has an affinity to cocaine receptors. Intranasal administration of GNC 92H2-pVIII (1.0 × 1014) resulted in detection of the modified phage in the brain of rats and, as expected, the displayed protein has the ability to block the psychoactive effects of cocaine [240].
Carrera 等人（2004 年）制备了显示与可卡因受体有亲和力的 scFv GNC 92H2 的噬菌体。给大鼠鼻内注射 GNC 92H2-pVIII（1.0 × 10 14 ）后，可在大鼠大脑中检测到改良噬菌体，正如预期的那样，显示的蛋白质具有阻断可卡因精神作用的能力[ 240]。
All these approaches enhance the detection or specificity of targeting and may be useful in creating bacteriophages for biomedical purposes, both diagnostic and therapeutic.
所有这些方法都增强了靶向的检测或特异性，可能有助于制造用于诊断和治疗的生物医学目的的噬菌体。