RGD环肽修饰白蛋白-三氧化二砷靶向胰腺癌肿瘤微环境的机制研究

Mechanistic study of RGD cyclic peptide-modified albumin-arsenic trioxide targeting the pancreatic cancer tumor microenvironment

关键词:  三氧化二砷  人血清白蛋白  精氨酸-甘氨酸-天冬氨酸  胰腺癌  巨胞饮

Key words:  Arsenic trioxide  Human serum albumin  Arginine-glycine-aspartic acid  Pancreatic cancer  Macropinocytosis.

摘要:

目的 构建精氨酸-甘氨酸-天冬氨酸（Arg-Gly-Asp，RGD）环肽修饰人血清白蛋白（Human serum albumin，HSA）耦合三氧化二砷（Arsenic trioxide，ATO）纳米药物，对其理化性质进行表征，并研究其体外抗胰腺癌药效和在胰腺肿瘤中的转运作用。方法 通过砷硫键将HSA与ATO进行结合构建三氧化二砷白蛋白（HSA-ATO）纳米药物，进一步在表面修饰RGD构建RGD-HSA-ATO纳米药物。分别使用透射电子显微镜、马尔文粒径仪检测HSA-ATO和RGD-HSA-ATO粒径、电位及PDI。使用电感耦合等离子体光谱仪（ICP）分别检测HSA-ATO和RGD-HSA-ATO的砷含量，计算纳米药物的载药量。进一步构建共培养AsPC-1 mcherry /HPSCCMFDA肿瘤球模型，使用HSA-ATO和RGD-HSA-ATO处理肿瘤球模型，考察纳米药物的体外药效和转运。使用转运抑制剂考察肿瘤球摄取RGD-HSA-ATO的作用机制。结果 研究显示成功构建HSA-ATO和RGD-HSA-ATO纳米药物。透射电镜和粒径检测结果显示HSA-ATO和RGD-HSA-ATO粒径均一，分布均匀，粒径分别为33.20±1.80nm和34.80±2.30nm，呈负电性。ICP结果显示HSA-ATO和RGD-HSA-ATO的载药量分别为3.00±0.04%和2.99±0.06%，体外释放具有pH和谷胱甘肽（glutathione，GSH）响应性。体外共培养模型结果显示RGD和HAS修饰显著增强了药物在肿瘤球内的转运以及抗肿瘤药效。RGD-HSA-ATO干预144 h后，肿瘤细胞存活率仅为57.33±1.88%，显著低于ATO处理组。转运机制研究结果显示RGD-HSA-ATO通过巨胞饮途径转运入肿瘤球。结论 RGD-HSA-ATO显著增强了ATO在胰腺癌细胞中的转运和药效，为中药亲水性小分子抗肿瘤药物难以突破胰腺癌致密间质渗透屏障到达肿瘤组织深部发挥药效提供研究基础。

Abstract:

ABSTRACT: OBJECTIVE To construct an arginine-glycine-aspartic acid (RGD) peptide-modified human serum albumin (HSA) arsenic trioxide (ATO) nanomedicine, characterize its physicochemical properties, and evaluate its in vitro anti-pancreatic cancer efficacy and transport mechanisms. METHODS HSA was conjugated with arsenic trioxide (ATO) via arsenic-sulfur bonds to form HSA-ATO, which was further modified with RGD peptides to create RGD-HSA-ATO. The particle size, zeta potential, and polydispersity index (PDI) of HSA-ATO and RGD-HSA-ATO were assessed using transmission electron microscopy (TEM) and malvern particle size analyzer. The arsenic content in HSA-ATO and RGD-HSA-ATO was determined using an inductively coupled plasma spectrometer (ICP), and the drug loading capacity was calculated. An in vitro co-culture AsPC-1 mcherry/HPSCCMFDA tumor spheroid model was established, and the efficacy and transport of the nanomedicines were evaluated by treating with HSA-ATO and RGD-HSA-ATO, with ATO as a control. Further, the transport mechanism of RGD-HSA-ATO uptake by tumor spheroids was investigated using transport inhibitors. RESULTS HSA-ATO and RGD-HSA-ATO nanomedicines were successfully constructed. TEM imaging showed uniform particle sizes with good distribution, measuring 33.20 ± 1.80 nm and 34.80 ± 2.30 nm, respectively, and both showed negative zeta potentials. ICP results revealed drug loading capacities of 3.00 ± 0.04% for HSA-ATO and 2.99 ± 0.06% for RGD-HSA-ATO. In vitro release studies showed pH and glutathione (GSH) responsiveness. In the co-culture model, RGD and HSA modifications significantly enhanced drug transport within the tumor spheroids. After 144 hours of RGD-HSA-ATO treatment, the tumor cell viability was 57.33 ± 1.88%, significantly lower than the ATO-treated group. Transport mechanism studies indicated that RGD-HSA-ATO entered tumor cells via macropinocytosis. CONCLUSION RGD-HSA-ATO significantly enhanced the transport of ATO into pancreatic cancer cells. This study provides a foundation for overcoming the dense stromal barrier in pancreatic cancer, allowing hydrophilic small molecule anticancer drugs to penetrate deeper tumor tissues and exert their therapeutic effects.