目的 制备血小板衍生生长因子受体β(platelet-derived growth factor receptor β,PDGFRβ)靶向性分子探针,并评价其用于肺癌光学分子影像的可能性.方法 通过基因工程方法,利用大肠杆菌重组表达系统生产并制备PDGFRβ特异性亲和体Z-tri.利用流式细胞术分析Z-tri对体外培养细胞的结合能力,用示踪法监测Z-tri在移植瘤内的细胞分布.将近红外荧光染料CF750与Z-tri偶联制备光学分子探针CF750-Z-tri,并利用光学成像系统检测其对人肺癌细胞移植瘤的显像效果.结果 PDGFRβ特异性亲和体Z-tri在大肠杆菌中获得大量表达,且能通过简单的亲和层析进行纯化.该蛋白体外条件下能与PDGFRβ阳性细胞结合,进入移植瘤内也主要分布于PDGFRβ强阳性细胞.近红外荧光染料CF750能够高效地与Z-tri偶联.制备的近红外荧光探针CF750-Z-tri能快速、清楚地通过光学成像显示肺癌移植瘤.结论 近红外光学分子探针CF750-Z-tri能够用于肺癌分子影像,在利用术中导航指导肺癌组织精准切除方面有应用前景.

Current understanding of toxicity mechanisms of nanoparticles is still far from comprehensive, partly because of the neglect of control factors such as the dependence of mechanism activation on the exposure dosage and particle size. To reveal molecular mechanisms of silver nanoparticle (AgNP) toxicity, the model ciliate Paramecium multimicronucleatum was exposed for 12 h to different concentrations of AgNPs with particle size of 20 nm (0.08, 0.12, and 0.30 mg/l) and 40 nm (0.08 and 0.30 mg/l). Transcriptomes of the tested ciliates were then analyzed based on dendrograms of gene expression, Gene Ontology (GO) terms, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, and up- and down-regulated genes. Results showed that: (1) toxicity mechanisms of AgNP revealed by analyses of GO and KEEG were significantly involved in the metabolic pathways of nutrients and the biosynthesis of macromolecules; (2) the top five up-regulated genes were mainly related to biological oxidation, biosynthesis, and oxidative stress, while top five down-regulated genes were mainly related to glycolysis; (3) activated mechanisms varied both in quantity and in type with dosages and particle sizes of AgNPs; (4) AgNP-treatments with different exposure dosages and particle sizes can produce the same toxicity in terms of 12 h-EC50, but the underlying molecular mechanisms differed significantly. In brief, this study provides insights into the molecular mechanisms of AgNP toxicity through transcriptome analyses and confirmed their dependence of activation on the exposure dosage and particle size of AgNPs.

Immunotherapies based on immune checkpoint-blocking antibodies have been considered the most attractive cancer treatments in recent years. However, the systemic administration of immune checkpoint-blocking antibodies is limited by low response rates and high risk of inducing immune-related adverse events (irAEs), which might be overcome by the tumor-targeted delivery of these antibodies. To achieve tumor-targeted delivery, immune checkpoint-blocking antibodies are usually modified with tumor-homing ligands through difficult genetic fusion or chemical conjugation. As most immune checkpoint-blocking antibodies are immunoglobin G (IgG) antibodies, we hypothesize that these IgG antibodies might be noncovalently modified with a tumor-homing ligand fused to an IgG-binding domain (IgBD). To test this hypothesis, the tumor-homing ZPDGFRβ affibody, which targets platelet-derived growth factor receptor β (PDGFRβ), was fused to the Fab-selective IgBD in a trimeric format. After mixing ZPDGFRβ fused to the IgBD with immune checkpoint-blocking IgG against programmed death-ligand 1 (αPD-L1), a novel homogenous complex was formed, indicating that αPD-L1 had been successfully modified with ZPDGFRβ fused to the IgBD. ZPDGFRβ-modified αPD-L1 bound to both PDGFRβ and PD-L1, thus leading to greater tumor uptake and antitumor effects in mice bearing PDGFRβ+PD-L1+ tumor grafts. In addition, due to the broad spectrum of IgBD for IgG, immune checkpoint-blocking IgG antibodies against cytotoxic T-lymphocyte-associated protein 4 (αCTLA-4) and signal regulatory protein alpha (αSIRPα) were also modified with ZPDGFRβ fused to the IgBD. These results demonstrated that a tumor-homing ligand fused to the IgBD might be developed as a versatile platform for the modification of immune checkpoint-blocking IgG antibodies to achieve tumor-targeted delivery.

Due to their potent immune stimulation, tumor necrosis factor alpha (TNFα) variants with tumor-homing activity are attractive as novel antitumor drugs. The promising antitumor effect of NGR-TNFα in clinical trials triggered extensive interest in developing novel tumor-homing TNFα variants in recent years. Owing to its promising antitumor effect, NGR-TNFα is usually used as a control for newly developed tumor-homing TNFα variants. In our previous works, we produced a pericyte-targeting Z-TNFα at high levels using the Escherichia coli (E. coli) M15-pQE30 system. To further compare Z-TNFα and NGR-TNFα, we attempted to express NGR-TNFα using the same system. Surprisingly, native NGR-TNFα was expressed at a low (~ 0.2 mg/L) level in E. coli M15 containing the pQE30 plasmid. However, a single nucleotide mutation of C to G, resulting in a substitution of leucine (L) with valine (V) at the start of TNFα, increased the expression of NGR-TNFα by ~ 100 times through improving transcription. In addition, the amino acid substitution showed a little impact on the receptor binding, in vitro cytotoxicity, and in vivo antitumor effect of NGR-TNFα. As fusing NGR to the N-terminus of TNFα with a valine substitution did not reduce the protein yield, the TNFα gene with a C > G mutation might be used to prepare novel tumor-homing TNFα when the native TNFα-based variant is expressed at an extremely low level in E. coli. Notably, in addition to the mutated valine, the impact of N-terminal additional amino acids provided by pQE30 vector on the function of TNFα variant must be carefully evaluated. • A single nucleotide mutation increased the expression of NGR-TNFα by two orders. • Nucleotide mutation-induced amino acid substitution did not reduce NGR-TNFα activity.