L. E. J. Nanotechnology Center, H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan.
L. E. J. Nanotechnology Center, H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan.
Int J Pharm. 2023 Aug 25;643:123227. doi: 10.1016/j.ijpharm.2023.123227. Epub 2023 Jul 13.
We have synthesized new lipidic prodrugs of diclofenac by grafting aliphatic chains (C10, C12, C16 and C18) to diclofenac through an ester bond. Their molecular formulas were confirmed through HR-MS and the formation of ester bond by FTIR and NMR spectroscopy. Nanoparticles of the different prodrugs were successfully formulated using emulsion evaporation method and DSPE-PEG as the only excipient. All nanoparticles were spherical and had a size between 110 and 150 nm, PdI ≤ 0.2 and negative Zeta potential values from -30 to -50 mV. In addition, they were stable upon storage at 4 °C up to 30-35 days. The encapsulation efficiency of the prodrug was above 90 % independently of the aliphatic chain length grafted. Nanoparticles did not induce any toxicity on LPS-activated THP-1 cells up to a concentration of 100 μg/mL (equivalent diclofenac) whereas diclofenac sodium salt IC was around 20 μg/mL. Following incubation of nanoparticles with LPS-activated THP-1 cells, a dose dependent inhibition of TNF-α was observed comparable to standard diclofenac sodium. Based on in vitro studies representative nanoparticles, Prodrug 3 NPs (C16 aliphatic chain) were selected for further in vitro and in vivo studies. Upon incubation in murine plasma, Prodrug 3 NPs underwent an enzymatic cleavage and almost 70 % of diclofenac was released from nanoparticles in 8 h. In vivo studies on a collagen induced arthritis murine model showed contrasted results: on one hand Prodrug 3 NPs led to a significant decrease of arthritis score and of paw volume compared to PBS after the second injection, on the other hand the third injection induced an important hepatic toxicity with the death of half of the mice from the NP group. To promote the reduction of inflammation while avoiding hepatic toxicity using NPs would require to precisely study the No Observable Adverse Effect Level and the schedule of administration in the future.
我们通过酯键将脂肪链（C10、C12、C16 和 C18）接枝到双氯芬酸上，合成了双氯芬酸的新型脂质前体药物。通过高分辨质谱（HR-MS）确认了它们的分子式，并通过傅里叶变换红外光谱（FTIR）和核磁共振光谱（NMR）确认了酯键的形成。通过乳化蒸发法成功地将不同前体药物的纳米粒进行了制剂化，并用 DSPE-PEG 作为唯一的赋形剂。所有的纳米粒均为球形，粒径在 110 至 150nm 之间，PdI≤0.2，Zeta 电位值为负 30 至-50mV。此外，它们在 4°C 下储存 30-35 天也很稳定。前体药物的包封效率均高于 90%，与接枝的脂肪链长度无关。纳米粒在 100μg/mL（相当于双氯芬酸钠）的浓度下不会诱导 LPS 激活的 THP-1 细胞产生任何毒性，而双氯芬酸钠盐的 IC 约为 20μg/mL。将纳米粒与 LPS 激活的 THP-1 细胞孵育后，观察到 TNF-α 的抑制作用呈剂量依赖性，与标准双氯芬酸钠相当。基于体外研究，选择具有代表性的纳米粒，即前体药物 3 NPs（C16 脂肪链），进行进一步的体外和体内研究。在鼠血浆中孵育时，前体药物 3 NPs 经历了酶切，8 小时内几乎 70%的双氯芬酸从纳米粒中释放出来。在胶原诱导的关节炎鼠模型中的体内研究结果表明存在对比：一方面，与 PBS 相比，前体药物 3 NPs 在第二次注射后导致关节炎评分和爪体积显著降低；另一方面，第三次注射导致了重要的肝毒性，NP 组中有一半的小鼠死亡。为了使用 NPs 促进炎症的减轻，同时避免肝毒性，未来需要精确研究无明显不良反应水平（No Observable Adverse Effect Level，NOAEL）和给药方案。
