Zlotnikov Igor D, Ezhov Alexander A, Belogurova Natalya G, Kudryashova Elena V
Faculty of Chemistry, Lomonosov Moscow State University, Leninskie Gory, 1/3, 119991 Moscow, Russia.
Faculty of Physics, Lomonosov Moscow State University, Leninskie Gory, 1/2, 119991 Moscow, Russia.
Gels. 2024 Aug 30;10(9):567. doi: 10.3390/gels10090567.
Rhodamine 6G (R6G) and 4-nitro-2,1,3-benzoxadiazole (NBD) linked through a spacer molecule spermidine (spd), R6G-spd-NBD, produces a fluorescent probe with pH-sensitive FRET (Förster (fluorescence) resonance energy transfer) effect that can be useful in a variety of diagnostic applications. Specifically, cancer cells can be spotted due to a local decrease in pH (Warburg effect). In this research, we applied this approach to intracellular infectious diseases-namely, leishmaniasis, brucellosis, and tuberculosis, difficult to treat because of their localization inside macrophages. R6G-spd-NBD offers an opportunity to detect such bacteria and potentially deliver therapeutic targets to treat them. The nanogel formulation of the R6G-spd-NBD probe (nanoparticles based on chitosan or heparin grafted with lipoic acid residues, Chit-LA and Hep-LA) was obtained to improve the pH sensitivity in the desired pH range (5.5-7.5), providing selective visualization and targeting of bacterial cells, thereby enhancing the capabilities of CLSM (confocal laser scanning microscopy) imaging. According to AFM (atomic force microscopy) data, nanogel particles containing R6G-spd-NBD of compact structure and spherical shape are formed, with a diameter of 70-100 nm. The nanogel formulation of the R6G-spd-NBD further improves absorption and penetration into bacteria, including those located inside macrophages. Due to the negative charge of the bacteria surface, the absorption of positively charged R6G-spd-NBD, and even more so in the chitosan derivatives' nanogel particles, is pronounced. Additionally, with a pH-sensitive R6G-spd-NBD fluorescent probe, the macrophages' lysosomes can be easily distinguished due to their acidic pH environment. CLSM was used to visualize samples of macrophage cells containing absorbed bacteria. The created nanoparticles showed a significant selectivity to model vs. bacterial cells, and the R6G-spd-NBD agent, being a mild bactericide, cleared over 50% in conditions where remained almost unaffected. Taken together, our data indicate that R6G-spd-NBD, as well as similar compounds, can have value not only for diagnostic, but also for theranostic applications.
通过间隔分子亚精胺（spd）连接的罗丹明6G（R6G）和4-硝基-2,1,3-苯并恶二唑（NBD），即R6G-spd-NBD，产生了一种具有pH敏感荧光共振能量转移（FRET）效应的荧光探针，可用于多种诊断应用。具体而言，由于局部pH值降低（瓦伯格效应），癌细胞可以被检测到。在本研究中，我们将这种方法应用于细胞内感染性疾病，即利什曼病、布鲁氏菌病和结核病，这些疾病由于其在巨噬细胞内的定位而难以治疗。R6G-spd-NBD提供了检测此类细菌并潜在地提供治疗靶点以治疗它们的机会。获得了R6G-spd-NBD探针的纳米凝胶制剂（基于壳聚糖或接有硫辛酸残基的肝素的纳米颗粒，即壳聚糖-硫辛酸（Chit-LA）和肝素-硫辛酸（Hep-LA）），以提高在所需pH范围（5.5 - 7.5）内的pH敏感性，实现对细菌细胞的选择性可视化和靶向，从而增强共聚焦激光扫描显微镜（CLSM）成像的能力。根据原子力显微镜（AFM）数据，形成了结构紧凑、呈球形的含有R6G-spd-NBD的纳米凝胶颗粒，直径为70 - 100纳米。R6G-spd-NBD的纳米凝胶制剂进一步提高了对细菌的吸收和渗透能力，包括那些位于巨噬细胞内的细菌。由于细菌表面带负电荷，带正电荷的R6G-spd-NBD的吸收，在壳聚糖衍生物的纳米凝胶颗粒中表现得更为明显。此外，使用对pH敏感的R6G-spd-NBD荧光探针，可以很容易地分辨出巨噬细胞的溶酶体，因为它们的pH环境呈酸性。CLSM用于可视化含有被吸收细菌的巨噬细胞样本。所制备的纳米颗粒对模型细菌细胞与目标细菌细胞表现出显著的选择性，并且R6G-spd-NBD试剂作为一种温和的杀菌剂，在目标细菌几乎不受影响的情况下清除了超过50%的模型细菌。综上所述，我们的数据表明R6G-spd-NBD以及类似化合物不仅在诊断方面有价值，而且在治疗诊断应用方面也有价值。
