Unimolecular micelles based on hyperbranched polyamidoamine (PAMAM) dendrimer were synthesized as both a cargo delivery vector and an imaging agent for triple-negative breast tumors, and the chemical synthesis procedures are detailed in this study. With the chemical conjugation of a peptide (F3, against cellular nucleolin) to increase its cellular internalization, these micelles can accumulate potently and specifically in breast cancer cells (e.g., MDA-MB-231). The size and morphology of these PAMAM-based micelles have been measured by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The hydrazone bond (responsive to pH alteration) between the loaded doxorubicin (DOX, as a model drug here) and PAMAM micelles enables cargo release following pH changes. Flow cytometry and confocal fluorescence microscopy revealed that PAMAM micelles with F3 attachment (PAMAM-DOX-F3) had stronger internalization into MDA-MB-231 cells (nucleolin-positive) than PAMAM micelles without F3 conjugation (PAMAM-DOX), whereas both of them have minimal interactions with L929 fibroblasts (nucleolin-negative). The positron-emitting isotope ⁶⁴Cu was added into PAMAM micelles by chelation to track their pharmacokinetic behavior (organ distribution profile) in vivo by positron emission tomography (PET) imaging. Serial PET imaging demonstrated that the accumulation of ⁶⁴Cu-PAMAM-DOX-F3 in MDA-MB-231 tumors was fast, potent, and persistent (tumor uptake: 6.1 ± 1.2 %injection dose per gram [%ID/g] at 24 h p.i.), significantly higher than that of ⁶⁴Cu-PAMAM-DOX (2.5 ± 0.4 %ID/g at the same time). Their distribution profiles in other organs/tissues were quite similar, with a relatively short circulation time. In addition, ex vivo fluorescence imaging confirmed that DOX can be delivered efficiently by these PAMAM micelles to MDA-MB-231 tumors. Deducing from these data, we believe that PAMAM-based micelles can be useful for selective combinational treatment of cancer.

Statement of Significance

As a very useful biomaterial for theranostic purposes, one of the major hurdles for micelles (particularly those from self-assembling) is their relatively low stability, especially when administered in vivo. In this study we are trying to overcome this limitation by designing unimolecular micelles (based on the concept of “one micelle is composed by one molecule”) from polyamidoamine dendrimers (PAMAM) in which the drug cargos (e.g. doxorubicin) are chemically attached via a hydrazone bond, and use them as a tumor-selective diagnostic/therapeutic platform. These unimolecular micelles possess superior stability over conventional micelles, and can undertake stimulus (pH) – responsive cargo release for more “targeted” cancer therapy. With the incorporation of a tumor-targeting peptidic sequence (F3) and a positron emitting isotope (copper-64), the pharmacokinetic behavior of these micelles can be readily monitored by positron emission tomography (PET) imaging technique, to confirm their specificity against cancer tissues. With further optimization, this micellar platform can have a broad clinical applicability due to its biocompatibility, selectivity, and stability.

中文翻译：

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正电子发射层析成像可引导的单分子胶束纳米平台用于癌症治疗学的应用

合成了基于超支化聚酰胺酰胺（PAMAM）树状大分子的单分子胶束，既作为载药载体，又是三阴性乳腺肿瘤的显像剂，本研究详细介绍了化学合成步骤。通过肽（F3，针对细胞核仁素）的化学缀合以增加其细胞内在化作用，这些胶束可以有效地且特异性地在乳腺癌细胞中蓄积（例如，MDA-MB-231）。这些基于PAMAM的胶束的大小和形态已通过透射电子显微镜（TEM）和动态光散射（DLS）进行了测量。负载的阿霉素（此处为模型药物，DOX）和PAMAM胶束之间的hydr键（响应pH改变）可在pH值变化后释放货物。流式细胞仪和共聚焦荧光显微镜检查显示，具有F3附着作用的PAMAM胶束（PAMAM-DOX-F3）较不具有F3结合作用的PAMAM胶束（PAMAM-DOX）具有更强的内在化作用进入MDA-MB-231细胞（核仁素呈阳性）。它们与L929成纤维细胞（核仁素阴性）的相互作用极小。通过螯合将发射正电子的同位素⁶⁴ Cu添加到PAMAM胶束中，以追踪其药代动力学行为（器官分布图）在体内通过正电子发射断层扫描（PET）成像。连续PET成像显示，MDA-MB-231肿瘤中⁶⁴ Cu-PAMAM-DOX-F3的积累快速，有效且持久（肿瘤摄取：24克时每克[％ID / g]注射剂量为6.1±1.2％） h pi。），显着高于⁶⁴ Cu-PAMAM-DOX（同时为2.5±0.4％ID / g）。它们在其他器官/组织中的分布情况非常相似，循环时间相对较短。另外，离体荧光成像证实DOX可以被这些PAMAM胶束有效地递送至MDA-MB-231肿瘤。根据这些数据，我们认为基于PAMAM的胶束可用于选择性联合治疗癌症。

重要声明

作为用于治疗用途的非常有用的生物材料，胶束（尤其是自组装胶束）的主要障碍之一是其相对较低的稳定性，尤其是在体内给药时。在这项研究中，我们试图通过设计聚酰胺酸树枝状聚合物（PAMAM）的单分子胶束（基于“一个分子由一个分子组成”的单分子胶束）来克服这一局限，其中药物货物（例如阿霉素）通过键，并将其用作肿瘤选择性诊断/治疗平台。这些单分子胶束比常规胶束具有更高的稳定性，并且可以进行刺激（pH）的反应性货物释放，以进行更“有针对性”的癌症治疗。结合肿瘤靶向肽序列（F3）和正电子发射同位素（copper-64），这些胶束的药代动力学行为可以通过正电子发射断层扫描（PET）成像技术轻松监测，以确认其对癌症组织的特异性。通过进一步优化，由于其生物相容性，选择性和稳定性，该胶束平台可具有广泛的临床适用性。