近红外光激发功能化上转换纳米颗粒用于解聚Aβ聚集体

doi:10.6023/A21050194

摘要/Abstract

阿尔兹海默症(AD)是一种进行性神经退行性疾病, 其特征是记忆力减退、神志不清和各种认知障碍. β-淀粉样蛋白(Aβ)的自组装聚集是阿尔茨海默症患者大脑的主要特征之一, 其加剧了AD患者的神经病变和认知障碍, 因此抑制Aβ聚集是一种潜在的治疗AD的策略. 光动力疗法是抑制Aβ聚集和解聚Aβ聚集体的有效方法. 然而, 大多数光敏剂为紫外和可见光激发, 在生物组织中的渗透深度低, 并引起严重的组织损伤, 这限制了其在生物医学中的应用. 本工作构建了一种近红外光激发的双靶向上转换纳米体系应用于抑制Aβ聚集过程. 以核壳结构的上转换纳米颗粒(UCNPs)作为光转换器, 通过两亲聚合物二硬脂酰基磷脂酰乙醇胺-聚乙二醇-马来酰亚胺(DSPE-PEG-MAL)的疏水包覆作用负载光敏剂二氢卟吩e6 (Ce6), 在纳米颗粒表面通过马来酰亚胺与巯基的特异性反应修饰了可跨越血脑屏障的肽链TGN和靶向Aβ₄₂的肽链QSH. 实验结果表明在近红外光激发下UCNPs通过发光共振能量转移(LRET)将能量转移至光敏剂Ce6, 使其跃迁至激发态后与周围的氧气分子作用产生单线态氧(¹O₂), 不可逆地氧化Aβ, 从而有效解聚了Aβ聚集体, 降低了Aβ聚集体的神经毒性. 此外, 该体系不仅具有良好的生物相容性, 而且可以有效穿过血脑屏障靶向作用于Aβ₄₂, 显示出其在活体水平治疗AD的潜力.

关键词: 近红外光, 上转换纳米颗粒, 光动力治疗, β-淀粉样蛋白, 阿尔兹海默症

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by memory loss, confusion, and a variety of cognitive disabilities. The self-assembly and aggregation of β-amyloid (Aβ) peptides is a main feature of the brain of Alzheimer's disease, which can aggravate the nerve damage and cognitive impairment of AD patients. Therefore, inhibition of the aggregation of Aβ peptides is recognized as a potential strategy to alleviate AD. Photodynamic therapy (PDT) is a creative method that has wide applications in suppressing amyloid aggregation or eliminating the amyloid aggregates. However, most photosensitizers are excited by ultraviolet and visible light, have a low penetration depth in biological tissues, and cause serious tissue damage, which limits their application in biomedicine. Herein, we report a dual-targeting upconversion nanoprobe excited by near-infrared light to inhibit the Aβ aggregation process. The core-shell structured upconversion nanoparticles NaYF₄:Yb,Er,Gd,Tm@NaYF₄ are used as light converters, and the photosensitizer chlorin-e6 (Ce6) is loaded through the hydrophobic coating of the amphiphilic polymer 1,2-distearoylsn-glycero-3-phosphoethanolamine-N- [maleimide(polyethyleneglycol)-2000] (DSPE-PEG-MAL). A blood-brain barrier targeting peptide, TGN, and a Aβ target peptide, QSH, are simultaneously modified on the surface of nanoparticles via the specific reaction between maleimide and sulfhydryl groups. The results indicate that UCNPs can transfer the excited-state energy to the photosensitizer Ce6 through luminescence resonance energy transfer (LRET) process under the excitation of near-infrared light irradiation. After transition to the excited state, Ce6 further interact with the surrounding oxygen molecules to produce singlet oxygen (¹O₂) and irreversibly oxidize β-amyloid, thus effectively disassembling Aβ aggregates and reducing the cytotoxicity associated with Aβ. In addition, UCNPs-Ce6-TQ not only exhibits good biocompatibility, but also can effectively cross the blood-brain barrier and target Aβ₄₂. This nanoprobe may be a powerful tool to inhibit the accumulation of Aβ in the brain and alleviate the neurotoxic damage caused by Aβ.

Key words: near infrared, upconversion nanoparticles, photodynamic therapy, beta-amyloid, Alzheimer's disease

引用此文

黄菊, 李贞, 刘志洪. 近红外光激发功能化上转换纳米颗粒用于解聚Aβ聚集体[J]. 化学学报, 2021, 79(8): 1049-1057.

Ju Huang, Zhen Li, Zhihong Liu. Functionalized Upconversion Nanoparticles for Disassembly of β‑Amyloid Aggregation with Near-Infrared Excitation[J]. Acta Chimica Sinica, 2021, 79(8): 1049-1057.

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图/表 6

图1. 基于双靶向负载光敏剂的上转换纳米体系在近红外光激发下抑制Aβ聚集的机理图. 在近红外光(980 nm)照射下, 上转换纳米颗粒发射655 nm可见光从而激发光敏剂Ce6产生单线态氧(1O2)不可逆地氧化了Aβ, 从而有效解聚Aβ聚集体

Figure 1. The mechanism diagram of the upconversion nanoparticles with dual-targeting and loaded photosensitizer to inhibit Aβ aggregation under near-infrared light irradiation. Under near-infrared light (980 nm) excitation, the upconversion nanoparticles emit 655 nm visible light to excite the photosensitizer Ce6 to produce singlet oxygen (1O2) to irreversibly oxidize Aβ peptides, thereby effectively disassembling Aβ aggregates

图2. (a) NaYF4:Yb,Er,Gd,Tm和(b) NaYF4:Yb,Er,Gd,Tm@NaYF4纳米颗粒的透射电镜图. (c) NaYF4:Yb,Er,Gd,Tm和(d) NaYF4:Yb,Er,Gd,Tm@NaYF4的粒径分布图

Figure 2. Transmission electron microscope images of (a) NaYF4:Yb,Er,Gd,Tm and (b) NaYF4:Yb,Er,Gd,Tm@NaYF4. Histograms of the size distribution of (c) NaYF4:Yb,Er,Gd,Tm and (d) NaYF4:Yb,Er,Gd,Tm@NaYF4

图3. (a) 光敏剂Ce6的紫外吸收谱图与水相UCNPs-PEG在近红外光照下上转换发光谱图重叠. (b) UCNPs负载光敏剂Ce6前后上转换发光光谱. (c) 负载不同浓度Ce6 (0~110 μg/mL)的UCNPs (1 mg/mL)的UV-vis吸收光谱. (d) UCNPs-Ce6-TQ的紫外吸收图与光敏剂以及染料标记的两条肽链的紫外吸收对比

Figure 3. (a) The UV absorption spectrum of the photosensitizer Ce6 overlaps with the upconversion emission spectrum of UCNPs-PEG in aqueous under near-infrared light excitation. (b) The upconversion luminescence spectra of UCNPs before and after loading the photosensitizer Ce6. (c) UV-vis absorbance spectra of UCNPs (1 mg/mL) loaded with different concentrations (0~110 μg/mL) of Ce6. (d) Comparison between the UV-vis absorption spectra of UCNPs-Ce6-TQ, photosensitizer and the two peptide chains labeled with the dyes

图4. UCNPs-Ce6-TQ在体外光氧化抑制Aβ聚集体. (a) 480 nm处ThT的荧光强度. (b) Aβ肽和与UCNPs-Ce6-TQ共孵育的Aβ肽在黑暗或近红外光照条件下的圆二色光谱. (c~e) 天然Aβ肽(20 μmol/L)和与UCNPs-Ce6-TQ共孵育的Aβ肽在不同条件下所得到的AFM图像. (f, g) 天然Aβ肽和与UCNPs-Ce6-TQ共孵育的Aβ肽在近红外光照后的DLS粒径分布图

Figure 4. UCNPs-Ce6-TQ photooxidation inhibits Aβ aggregates in vitro. (a) Fluorescence intensity of ThT at 480 nm. (b) Circular dichroism spectrum of natural Aβ peptides and Aβ peptides co-incubated with UCNPs-Ce6-TQ under dark or near-infrared light conditions. (c~e) AFM images of natural Aβ peptides (20 μmol/L) and Aβ peptides co-incubated with UCNPs-Ce6-TQ in the dark or under near-infrared light conditions. DLS particle size distribution of (f) natural Aβ peptide and (g) Aβ peptide co-incubated with UCNPs-Ce6-TQ under near-infrared light irradiation

图5. (a) 体外血脑屏障模型的示意图. (b) UCNPs-Ce6和UCNPs-Ce6-TQ的体外BBB渗透性

Figure 5. (a) Scheme showing the design of the blood-brain barrier model. (b) In vitro BBB permeability of UCNPs-Ce6 and UCNPs-Ce6-TQ

图6. 使用MTT分析评估UCNPs-Ce6-TQ的生物相容性和近红外光激发下UCNPs-Ce6-TQ对Aβ肽诱导的细胞毒性的抑制作用. (a) 不同浓度 (0、0.1、0.2、0.3、0.4和0.5 mg/mL) UCNPs-Ce6-TQ与PC12细胞孵育后细胞存活率分析. (b) 天然Aβ肽与用不同浓度UCNPs-Ce6-TQ处理的Aβ肽在黑暗或近红外光照条件下对PC12细胞活力的影响

Figure 6. MTT analysis to evaluate the biocompatibility of UCNPs-Ce6-TQ and the inhibition of Aβ peptide-induced cytotoxicity under near-infrared light excitation. (a) Cell viability analysis of UCNPs-Ce6-TQ incubated with PC12 cells at different concentrations (0, 0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL.) (b) Cell viability of PC12 incubated with natural Aβ peptides and Aβ peptides treated with different concentrations of UCNPs-Ce6-TQ under dark or near-infrared light irradiation conditions

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