Journal of Nanoparticle Research ( IF 2.1 ) Pub Date : 2021-08-24 , DOI: 10.1007/s11051-021-05296-0 Mehreen Rahman 1, 2 , Ummarah Kanwal 1 , Uzma Azeem Awan 1 , Abida Raza 1 , Jamshaid Ali Khan 2
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Methotrexate (MTX) is a well-known chemotherapeutic agent for solid tumor. However, its clinical usage is limited due to low permeability, cellular drug efflux, and non-specific drug delivery. These constraints require dose dumping and result in dose-dependent toxic effects. In the current study, MTX-loaded PEGylated gold nanoparticles (PEG-MTX-AuNPs) exposed to acidic pH (tumor cell) trigger a significant drug release profile. MTX was conjugated to citrate functionalized AuNP and stabilized by thiolated methyl polyethylene glycol (mPEG-SH). PEG-MTX-AuNP was cationic with 39.5 ± 1.2 mV zeta potential and a particle size of 39 nm, as revealed by DLS and TEM. AuNP size ranging from 40 to 50 nm was favorable for maximum endosomal uptake and fast drug release. MTX-AuNP showed 39.4% (22.28 µg/ml) drug loading efficiency. Spectroscopic examinations confirmed MTX chemisorption onto AuNPs via carboxyl (–COOH) and gold dative bond. In vitro release study indicated a 6.5-fold increased MTX release in the acidic environment (pH 4.5) compared to physiological pH. The terminal mPEG provided stability in a biological medium, refrained from protein deactivation, and exhibited significant hemocompatibility (less than 5% hemolysis up to 10 μM concentration). In vitro cytotoxicity against human rhabdomyosarcoma (RD) cells indicated significant (p < 0.0001) anticancer activity of PEG-MTX-AuNPs with low median growth inhibition concentration (GI50 = 3.61 ± 0.94 μM) compared to MTX (13.12 ± 0.98 μM) and AuNPs (5.24 ± 0.98 μM) alone. In conclusion, PEG-MTX-AuNPs offer higher stability in ionic and biological media, hemocompatibility, and acidic pH-dependent MTX release, and mediate enhanced in vitro anticancer activity that makes them a potential candidate for skeletal muscle sarcomas.
Graphic abstract
Methotrexate (MTX) is a weak dicarboxylic acid which become ionized at pH 7.4 (PBS) and unionized at endosomal pH 4.5. In ionized state MTX exchanges the negatively charged citrate ions and chemisorbed on AuNPs surface to stabilize and cap AuNPs. While in acidic media MTX become unionized and released in media. The enhanced intracellular release improves therapeutic outcome of MTX. The chemisorption of MTX was further confirmed with FTIR, supported by XRD and UV results.
中文翻译：
载有甲氨蝶呤的聚乙二醇化金纳米颗粒作为血液相容性和 pH 响应性抗癌药物纳米偶联物
甲氨蝶呤 (MTX) 是众所周知的实体瘤化疗药物。然而，由于低渗透性、细胞药物外排和非特异性药物递送，其临床应用受到限制。这些限制需要剂量倾销并导致剂量依赖性毒性作用。在目前的研究中，负载 MTX 的聚乙二醇化金纳米粒子 (PEG-MTX-AuNPs) 暴露于酸性 pH 值（肿瘤细胞）会触发显着的药物释放曲线。MTX 与柠檬酸官能化的 AuNP 结合，并由硫醇化甲基聚乙二醇 (mPEG-SH) 稳定。PEG-MTX-AuNP 是阳离子，具有 39.5 ± 1.2 mV zeta 电位和 39 nm 的粒径，如 DLS 和 TEM 所示。40 到 50 nm 的 AuNP 尺寸有利于最大内体吸收和快速药物释放。MTX-AuNP 的载药效率为 39.4% (22.28 µg/ml)。光谱检查证实了 MTX 通过羧基 (–COOH) 和金配价键化学吸附到 AuNPs 上。体外释放研究表明，与生理 pH 值相比，酸性环境 (pH 4.5) 中 MTX 的释放增加了 6.5 倍。末端 mPEG 在生物介质中提供稳定性，避免蛋白质失活，并表现出显着的血液相容性（在 10 μM 浓度下溶血率低于 5%）。对人横纹肌肉瘤 (RD) 细胞的体外细胞毒性表明显着（并表现出显着的血液相容性（在 10 μM 浓度下溶血率低于 5%）。对人横纹肌肉瘤 (RD) 细胞的体外细胞毒性表明显着（并表现出显着的血液相容性（在 10 μM 浓度下溶血率低于 5%）。对人横纹肌肉瘤 (RD) 细胞的体外细胞毒性表明显着（p < 0.0001) PEG-MTX-AuNPs 的抗癌活性与 单独的 MTX (13.12 ± 0.98 μM) 和 AuNPs (5.24 ± 0.98 μM) 相比，具有低中值生长抑制浓度 (GI 50 = 3.61 ± 0.94 μM)。总之，PEG-MTX-AuNPs 在离子和生物介质中具有更高的稳定性、血液相容性和酸性 pH 依赖性 MTX 释放，并介导增强的体外抗癌活性，使其成为骨骼肌肉瘤的潜在候选者。
图形摘要
甲氨蝶呤 (MTX) 是一种弱二元羧酸，在 pH 7.4 (PBS) 时电离，在内体 pH 4.5 时电离。在电离状态下，MTX 交换带负电荷的柠檬酸根离子并化学吸附在 AuNPs 表面以稳定和覆盖 AuNPs。而在酸性介质中 MTX 会被结合并在介质中释放。增强的细胞内释放改善了 MTX 的治疗效果。FTIR 进一步证实了 MTX 的化学吸附，XRD 和 UV 结果支持。
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