Objective

One of the vital signaling pathways in cancer development and metastasis is mitogen-activated protein kinases (MAPKs). Bacillus anthracis Lethal Toxin (LT) is a potent MAPK signaling inhibitor. This toxin is comprised of two distinct domains, Lethal Factor (LF), MAPK inhibitor, and Protective Antigen (PA). To enter various cell lines, LF must be associated with the protective antigen (PA), which facilitates LF delivery. In the current study, to block MAPK signaling, LF was loaded into anti-CD19 immunoliposomes nanoparticle to deliver the cargo to Raji B cells.

Methods

The liposome nanoparticle was prepared using classical lipid film formation, then conjugated to anti-CD19 VHH. The binding efficiency was measured through flow cytometry. The targeted cytotoxicity of LF immunoliposome was confirmed by BrdU lymphoproliferation assay. This was followed by Real-Time PCR to assess the effect of formulation on pro-apoptotic genes. The inhibitory effect of LF on MAPK signaling was confirmed by western blot.

Results

Liposome nano-formulation was optimized to reach the maximum LF encapsulation and targeted delivery. Next, phosphorylation of MAPK pathway mediators like MEK1/2, P38 and JNK were inhibited following the treatment of Raji cells with LF-immunoliposome. The treatment also upregulated caspase genes, clearly illustrating cell death induced by LF through pyroptosis and caspase-dependent apoptosis.

Conclusions

In conclusion, anti-CD19 VHH immunoliposome was loaded with LF, a potent MAPK inhibitor targeting B cells, which curbs proliferation and ushers B cells toward apoptosis. Thus, immunoliposome presents as a versatile nanoparticle for delivery of LF to block aberrant MAPK activation. To use LF as a therapy, it would be necessary to materialize LF without PA. In the current study, PA was substituted with anti-CD19 immunoliposome to make it targeted to CD19⁺ while keeping the normal cells intact.

中文翻译：

---

基于脂质体的载有炭疽致死因子并配备抗CD19 VHH的纳米载体可有效抑制B细胞中的MAPK通路

镜片

癌症发展和转移的重要信号通路之一是丝裂原活化蛋白激酶 (MAPKs)。Bacillus anthracis Lethal Toxin (LT) 是一种有效的 MAPK 信号抑制剂。这种毒素由两个不同的结构域组成：致死因子 (LF)、MAPK 抑制剂和保护性抗原 (PA)。要进入各种细胞系，LF 必须与促进 LF 传递的保护性抗原 (PA) 结合。在目前的研究中，为了阻断 MAPK 信号传导，将 LF 加载到抗 CD19 免疫脂质体纳米颗粒中，以将货物输送到 Raji B 细胞。

方法

脂质体纳米颗粒是使用经典的脂质膜形成制备的，然后与抗 CD19 VHH 结合。通过流式细胞术测量结合效率。LF免疫脂质体的靶向细胞毒性通过BrdU淋巴增殖试验得到证实。随后进行实时 PCR 以评估制剂对促凋亡基因的影响。通过蛋白质印迹证实了LF对MAPK信号的抑制作用。

结果

脂质体纳米制剂经过优化，以达到最大的 LF 封装和靶向递送。接下来，在用 LF 免疫脂质体处理 Raji 细胞后，MAPK 通路介质如 MEK1/2、P38 和 JNK 的磷酸化受到抑制。该治疗还上调了半胱天冬酶基因，清楚地说明了LF通过细胞焦亡和半胱天冬酶依赖性细胞凋亡诱导的细胞死亡。

结论

总之，抗 CD19 VHH 免疫脂质体装载了 LF，这是一种针对 B 细胞的有效 MAPK 抑制剂，可抑制增殖并引导 B 细胞凋亡。因此，免疫脂质体作为一种多功能纳米颗粒呈现，用于递送 LF 以阻断异常 MAPK 激活。要将 LF 用作治疗，有必要在没有 PA 的情况下实现 LF。在目前的研究中，PA被抗CD19免疫脂质体取代，使其靶向CD19 ⁺，同时保持正常细胞完整。