CNR-ISMN, Istituto per lo Studio dei Materiali Nanostrutturati c/o Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali dell' Università di Messina , Viale F. Stagno d'Alcontres 31 , Messina 98166 , Italy.
Dipartimento di Area Medica , Università di Udine , P.le Kolbe 4 , Udine 33100 , Italy.
Biomacromolecules. 2019 Jul 8;20(7):2530-2544. doi: 10.1021/acs.biomac.9b00306. Epub 2019 Jun 25.
Nowadays, active targeting of nanotherapeutics is a challenging issue. Here, we propose a rational design of a ternary nanoassembly (SAP) composed of nonionic amphiphilic β-cyclodextrins (amphiphilic CD) incorporating pheophorbide (Pheo) as a phototherapeutic and an adamantanyl-folic acid conjugate (Ada-FA) to target tumor cells overexpressing α-folate receptor (FR-α(+)). Dynamic light scattering and ζ-potential pointed out the presence of nanoassemblies bearing a negative surface charge (ζ = -51 mV). Morphology of SAP was investigated by atomic force microscopy and microphotoluminescence, indicating the presence of highly emissive near-spherical assemblies of about 280 nm in size. Complementary spectroscopic techniques such as ROESY-NMR, UV/vis and steady-state fluorescence revealed that the folic acid protrudes out of amphiphilic CD rims, prone for recognition with FR-α. Pheo was strongly loaded in the nanoassembly mostly in monomeric form, thus generating singlet oxygen (O) and consequentely showing phototherapeutic action. SAP remained stable until 2 weeks in aqueous solutions. Stability studies in biologically relevant media pointed out the ability of SAP to interact with serum proteins by means of the oligoethylenglycole fringe, without destabilization. Release experiments demonstrated the sustained release of Pheo from SAP in environments mimiking physiological conditions (∼20% within 1 week), plausibly suggesting low Pheo leaking and high integrity of the assembly within 24 h, time spent on average to reach the target sites. Cellular uptake of SAP was confirmed by confocal microscopy, pointing out that SAP was internalized into the tumoral cells expressing FR-α more efficiently than SP. SAP showed improved phototoxicity in human breast MCF-7 cancer cells FR-α(+) (IC = 270 nM) with respect to human prostate carcinoma PC3 cells (IC = 700 nM) that express a low level of that receptor (FR-α(-)). Finally, an improved phototoxicity in FR-α(+) MCF-7 cells (IC = 270 nM) was assessed after treatment with SAP vs SP (IC = 600 nM) which was designed without Ada-FA as a targeting unit.
如今，主动靶向纳米治疗是一个具有挑战性的问题。在这里，我们提出了一种由非离子两亲性β-环糊精（两亲性 CD）组成的三元纳米组装体（SAP）的合理设计，该组装体包含作为光疗剂的原卟啉（Pheo）和靶向过度表达α-叶酸受体（FR-α(+）的金刚烷叶酸偶联物（Ada-FA）。动态光散射和 ζ-电位表明存在带有负表面电荷（ζ = -51 mV）的纳米组装体。通过原子力显微镜和微光致发光研究了 SAP 的形态，表明存在具有约 280nm 大小的高度发光近球形组装体。互补的光谱技术，如 ROESY-NMR、UV/vis 和稳态荧光表明，叶酸突出于两亲性 CD 边缘之外，易于与 FR-α 识别。Pheo 主要以单体形式强烈负载在纳米组装体中，从而产生单线态氧（O）并表现出光疗作用。SAP 在水溶液中稳定至 2 周。在生物相关介质中的稳定性研究表明，SAP 能够通过寡聚乙二醇边缘与血清蛋白相互作用，而不会发生不稳定。释放实验表明，在模拟生理条件的环境中，Pheo 从 SAP 中的持续释放（约 20%在 1 周内），可能表明 Pheo 泄漏低且组装体在 24 小时内完整性高，这是到达靶位点的平均时间。通过共聚焦显微镜证实了 SAP 的细胞摄取，指出 SAP 比 SP 更有效地被表达 FR-α 的肿瘤细胞内化。与表达低水平该受体（FR-α(-））的人前列腺癌 PC3 细胞（IC = 700 nM）相比，SAP 在人乳腺癌 MCF-7 癌细胞 FR-α(+）（IC = 270 nM）中显示出改善的光毒性。最后，与没有 Ada-FA 作为靶向单元设计的 SP（IC = 600 nM）相比，SAP 对 FR-α(+） MCF-7 细胞（IC = 270 nM）的光毒性得到了改善。
