Mouzoura Panagiota, Marazioti Antonia, Gkartziou Foteini, Metsiou Despoina-Nektaria, Antimisiaris Sophia G
Pharmaceutical Technology Laboratory, Department of Pharmacy, University of Patras, Rion, 26504, Greece.
Laboratory of Basic Sciences, Department of Physiotherapy, University of the Peloponnese, Sparti, 23100, Greece.
Int J Nanomedicine. 2025 Jan 7;20:239-265. doi: 10.2147/IJN.S494512. eCollection 2025.
FTY720 bioactive lipid has proliferative, osteoinductive, chemo attractive, and angiogenic properties, being thus a potential exogenous administered agent for promotion of bone regeneration. Herein we developed FTY720-loaded liposomes as a potential delivery system that could retain and prolong the bioactivity of the bioactive lipid and at the same time reduce its cytotoxicity (at high doses).
FTY720 liposomes were prepared by thin-lipid hydration and microfluidic flow focusing, and evaluated for their ability to induce proliferation, osteoinduction, and chemoattraction in three cell types: MC3T3-E1 pre-osteoblast cells, L929 fibroblast cells, and ATDC5 chondrogenic cells. The angiogenic activity of free and liposomal FTY720 was investigated using a chick chorioallantoic membrane assay. NBD-FTY720 cellular uptake was quantitated using flow cytometry and morphologically assessed by confocal microscopy. Implicated cellular signaling mechanisms were investigated by quantifying phosphorylated MAPK and CREB proteins.
FTY720 liposomes (~80-110 nm) with low polydispersity and ~100% loading were prepared using both methods. FTY720 demonstrated the ability to increase cell proliferation at 10-300nM doses but was cytotoxic at doses>400nM while the corresponding liposomal-FTY720 doses were non-cytotoxic, proving its reduced toxicity. In several cases (cells and doses), FTY720 liposomes demonstrated increased osteogenic differentiation of cells, proliferation, and migration compared to free FTY720, whereas both FTY720 forms demonstrated substantial angiogenic activity. Liposomal FTY720 cellular uptake was substantially higher than that of free FTY720 in some cases, a fact that may be connected to its higher bioactivity. Increased phosphorylated MAPK and CREB protein concentrations provided information about the potential cellular signaling mechanisms involved in FTY720-induced osteogenesis.
The current results confirm the high potential of FTY720 bioactive lipid, especially in its liposomal form, that demonstrated substantial reduction of cytotoxicity and prolonged preservation of the lipids bioactivity (compared to the free lipid), for accelerated treatment of bone defects. Interestingly, the current studies prove the potential of FTY720, especially in its liposomal form, to promote reprogramming of L929 fibroblasts into osteoblasts, a novel finding deserving future exploitation.
FTY720生物活性脂质具有增殖、骨诱导、化学吸引和血管生成特性，因此是一种促进骨再生的潜在外源性给药制剂。在此，我们开发了负载FTY720的脂质体作为一种潜在的递送系统，该系统可以保留并延长生物活性脂质的生物活性，同时降低其细胞毒性（高剂量时）。
通过薄脂质水合和微流控流动聚焦制备FTY720脂质体，并评估其在三种细胞类型中的诱导增殖、骨诱导和化学吸引能力：MC3T3-E1前成骨细胞、L929成纤维细胞和ATDC5软骨细胞。使用鸡胚绒毛尿囊膜试验研究游离和脂质体形式的FTY720的血管生成活性。使用流式细胞术对NBD-FTY720的细胞摄取进行定量，并通过共聚焦显微镜进行形态学评估。通过定量磷酸化的MAPK和CREB蛋白来研究相关的细胞信号传导机制。
两种方法均制备出了低多分散性（~80-110nm）且负载率约为100%的FTY720脂质体。FTY720在10-300nM剂量下具有增加细胞增殖的能力，但在剂量>400nM时具有细胞毒性，而相应的脂质体-FTY720剂量无细胞毒性，证明其毒性降低。在几种情况下（细胞和剂量），与游离FTY720相比，FTY720脂质体显示出细胞的成骨分化、增殖和迁移增加，而两种形式的FTY720均表现出显著的血管生成活性。在某些情况下，脂质体FTY720的细胞摄取显著高于游离FTY720，这一事实可能与其更高的生物活性有关。磷酸化的MAPK和CREB蛋白浓度的增加提供了有关FTY720诱导成骨潜在细胞信号传导机制的信息。
目前的结果证实了FTY720生物活性脂质的高潜力，特别是其脂质体形式，与游离脂质相比，其细胞毒性显著降低，脂质生物活性得以延长保存，可加速骨缺损的治疗。有趣的是，目前的研究证明了FTY720，特别是其脂质体形式，具有促进L929成纤维细胞重编程为成骨细胞的潜力，这是一个值得未来探索的新发现。
