Peptide motifs for insertion of radiolabeled biomolecules into cells and routing to the nucleus for cancer imaging or radiotherapeutic applications

Intracellular compartments, in particular the cytoplasm or nucleus, have generally been poorly accessible or inaccessible to radiolabeled biomolecules (e.g., monoclonal antibodies [mAbs], peptides, or oligonucleotides [ODNs]). However, recently cell-penetrating peptides (CPPs) and nuclear localizing peptide sequences (NLSs) have been shown to have the capability of inserting biomolecules into cells and transporting them to the cell nucleus. This discovery now presents intriguing new opportunities to design radiopharmaceuticals that could potentially probe, through imaging, the expression of key intracellular or intranuclear regulatory proteins that define the tumor phenotype, predict outcome, or act as sensitive reporters of response or resistance to treatment. CPPs could also more efficiently internalize radiolabeled antisense ODNs or peptide nucleic acids (PNAs) into tumor cells to enhance the sensitivity of imaging gene expression at the mRNA level. Perhaps one of the most exciting new developments to emerge is the use of NLS to route mAbs and peptides conjugated to nanometer-micrometer range Auger-electron-emitting radionuclides (e.g., (111)In) to the nucleus of cancer cells following their receptor-mediated internalization. In the nucleus, these electrons are highly potent in causing lethal DNA strand breaks. In some cases, NLSs are present naturally in peptide growth factors or their receptors, where they function to deliver internalized ligands to the nucleus, or alternatively, they can be introduced synthetically. This update reviews the properties of CPPs and NLS and focuses on their use for inserting radiolabeled biomolecules into cancer cells for imaging or targeted Auger electron radiotherapy of malignancies.     

Effect of the EGFR density of breast cancer cells on nuclear importation, in vitro cytotoxicity, and tumor and normal-tissue uptake of [111In]DTPA-hEGF

INTRODUCTION: Our objective was to evaluate the effect of epidermal growth factor receptor(s) (EGFR) density on the importation and nuclear localization of 111In-labeled diethylenetriaminepentaacetic acid human epidermal growth factor ([111In]DTPA-hEGF) in breast cancer (BC) cells in vitro and in tumor xenografts and normal tissues in vivo in athymic mice, as well as on its cytotoxicity and tumor and normal-tissue distribution. METHODS: The internalization and nuclear importation of [111In]DTPA-hEGF were measured in MCF-7, MDA-MB-231, BT-474 and MDA-MB-468 BC cells (10(4), 2 x 10(5), 6 x 10(5) and 10(6) EGFR/cell, respectively). The molecular size (Mr) distribution and immunoreactivity of nuclear radioactivity were characterized. Tumor and normal-tissue uptake of [111In]DTPA-hEGF in athymic mice implanted subcutaneously with BC xenografts were compared. Nuclear radioactivity in the tumor, lungs, liver, kidneys, spleen and colon was measured. RESULTS: There was a direct association between EGFR density and the nuclear localization of [111In]DTPA-hEGF in BC cells; nuclear importation approached saturation at 6 x 10(5) EGFR/cell. Almost all nuclear radioactivities exhibited an Mr of >100 kDa; immunoreactivity with anti-hEGF, anti-EGFR and anti-importin beta 1 antibodies was detected. The efflux of nuclear radioactivity was slowest for MDA-MB-468 cells. Cytotoxicity was correlated with EGFR expression. Uptake was greater in MDA-MB-468 than in MCF-7 xenografts and improved with preinjection of a 100-fold excess of unlabeled DTPA-hEGF. Nuclear importation was higher in liver, kidney and spleen cells than in tumor cells. CONCLUSION: [111In]DTPA-hEGF is translocated to the nucleus of BC cells complexed with EGFR and importin beta1. Nuclear importation and cytotoxicity are effected by EGFR density. The absence of hepatic and renal toxicities in [111In]DTPA-hEGF cannot be explained by a low efficiency of nuclear importation.     

<Superscript>123</Superscript>I-labeled HIV-1 tat peptide radioimmunoconjugates are imported into the nucleus of human breast cancer cells and functionally interact in vitro and in vivo with the cyclin-dependent kinase inhibitor,p21<Superscript>WAF-1/Cip-1</Superscript>

Purpose To evaluate the internalization and nuclear translocation of ¹²³I-tat-peptide radioimmunoconjugates in MDA-MB-468 breast cancer cells and their ability to interact with the cyclin-dependent kinase inhibitor, p21^WAF-1/Cip-1. Methods Peptides [GRKKRRQRRRPPQGYGC] harboring the nuclear-localizing sequence from HIV tat domain were conjugated to anti-p21^WAF-1/Cip-1 antibodies. Immunoreactivity was assessed by Western blot using lysate from MDA-MB-468 cells exposed to EGF to induce p21^WAF-1/Cip-1. Internalization and nuclear translocation were measured. The ability of tat-anti-p21^WAF-1/Cip-1 to block G₁-S phase arrest in MDA-MB-468 cells caused by EGF-induced p21^WAF-1/Cip-1 was evaluated. Tumor and normal tissue uptake were determined at 48 h p.i. in athymic mice implanted s.c. with MDA-MB-468 xenografts injected intratumorally with EGF. Results There was 13.4±0.2% of radioactivity internalized by MDA-MB-468 cells incubated with ¹²³I-tat-anti-p21^WAF-1/Cip-1 and 34.6±3.1% imported into the nucleus. Tat-anti-p21^WAF-1/Cip-1(8 μM) decreased the proportion of EGF-treated cells in G₁ phase from 81.9±0.7% to 46.1±0.7% (p<0.001), almost restoring the G₁ phase fraction to that of unexposed cells (25.8±0.2%). Non-specific tat-mouse IgG did not block EGF-induced G₁-S phase arrest. Tumor uptake of radioactivity was higher in mice injected with EGF to induce p21^WAF-1/Cip-1 than in mice not receiving EGF (3.1±0.4% versus 1.8±0.2% ID/g; p=0.04). Western blot analysis of tumors revealed a threefold increase in the p21^WAF-1/Cip-1/β-actin ratio. Conclusion We conclude that intracellular and nuclear epitopes in cancer cells can be functionally targeted with tat-radioimmunoconjugates to exploit many more epitopes for imaging and radiotherapeutic applications than have previously been accessible.     

Apoptotic epidermal growth factor (EGF)-conjugated block copolymer micelles as a nanotechnology platform for targeted combination therapy

The overexpression of epidermal growth factor receptor (EGFR) in human epithelial cancers has been associated with aggressive disease, poor patient prognosis, and a high incidence of metastases. In the present study, block copolymer micelles are conjugated with epidermal growth factor (EGF), which acts as both a targeting ligand for the drug carrier and an apoptotic factor against EGFR-overexpressing cancers. Drug-free EGF-conjugated micelles are shown to result in cell-cycle arrest at the G 1 phase and subsequent induction of cell-type-specific apoptosis in EGFR-overexpressing breast cancer cells as demonstrated by flow cytometric analysis. EGF delivered as EGF-conjugated micelles was found to be 13-fold more potent than free EGF; the IC 50 was decreased from 0.98 +/- 0.1 nM for free EGF to 0.076 +/- 0.01 nM for EGF micelles. The apoptotic micelles, however, are non-antiproliferative to cells expressing a low level of EGFR, suggesting that the apoptotic micelles have minimal or no toxicity against normal healthy tissues. Ellipticine, a chemotherapeutic agent, was loaded into the EGF-micelles after it had been shown, using the combination index-isobologram equation, to act synergistically with EGF. A 10-fold increase in EGF content in the ellipticine-loaded micelles lowered the IC 50 of ellipticine in EGFR-overexpressing breast cancer cells by more than 18-fold. The EGF-micelles have the potential to be further pursued as a versatile nanotechnology platform for targeted delivery of a wide range of chemotherapeutic agents as a combination therapy for the treatment of EGFR-overexpressing cancers.     

血浆置换法治疗重症肌无力患者的护理

采用血浆置换法治疗重症肌无力，在缓解肌无力症状方面，效果良好。笔者报告了９例重症肌无力患者７２次血浆置换治疗过程中的操作方法、术后常见并发症的防治措施以及出院指导。     

三级甲等医院急诊护士专业核心能力现状及相关因素调查

目的了解三级甲等医院急诊护士专业核心能力现状及相关因素,为探索急诊专科护士的培养模式提供依据。方法采用便利抽样法,应用自行设计的问卷对6所三级甲等综合性医院111名急诊护士进行调查。结果急诊护士自评专业核心能力得分(159.4±43.8)分;是否接受急救专科知识培训、培训时间及急救专科知识培训/学习方式不同者其自评核心能力得分比较,差异有统计学意义(均P0.01);急救专科知识培训时间是影响自评专业核心能力的主要因素(P0.01)。结论急诊护士专业核心能力欠佳,尤其缺乏科研及管理方面的能力;专科培训时间是主要影响因素。应针对性开展系统培训工作及设置合理的培训时间,以培养和提高急诊护士专业核心能力。     

Outcome of hospital deliveries of women living at high altitude: a study from Lhasa in Tibet

AIM: To describe rates of neonatal mortality, low birthweight (LBW), preterm birth and small for gestational age (SGA), and relate outcome to ethnicity and perinatal risk factors of liveborn infants of hospital deliveries in Lhasa. The differences in these variables between ethnic Tibetans and non-Tibetans were also studied. METHODS: Data were prospectively collected on the outcome of all liveborn infants born in four hospitals in the urban area of Lhasa, Tibet, in 2005. RESULT: A total of 2540 liveborn infants were recorded. The rates of LBW, preterm birth and SGA were 13.6%, 5.7% and 22.2%, respectively. Neonatal mortality rate was 42/1000 for the infants born alive in the hospitals. Lower GA, vaginal delivery, foetal distress and lack of prenatal care, but not ethnicity, were associated with increased risk of death in multivariate logistic regression. Tibetans had higher BW and lower rates of LBW, SGA, need of oxygen supplementation and maternal hypertension, but higher rates of foetal distress, caesarean section, multiple births and low Apgar scores. CONCLUSION: This study provided a profile of perinatal-neonatal care of hospital newborn infants in Lhasa, Tibet. The rates of neonatal mortality, LBW and SGA were high. The findings suggest ethnic differences in perinatal-neonatal adaptation to high altitude.     

Site-specific conjugation of HIV-1 tat peptides to IgG: a potential route to construct radioimmunoconjugates for targeting intracellular and nuclear epitopes in cancer

Purpose Our objective was to study the cellular and nuclear uptake of ¹²³I-mouse IgG (¹²³I-mIgG) linked to peptides [GRKKRRQRRRPPQGYGC] harbouring the membrane-translocating and nuclear import sequences of HIV-1 tat protein.Methods Carbohydrates on mIgG were oxidized by NaIO₄, then reacted with a 40-fold excess of peptides. Displacement of binding of anti-mouse IgG (Fab specific; -mFab) to ¹²³I-mIgG by tat-mIgG or mIgG was compared. Internalization and nuclear translocation of ¹²³I-tat-mIgG in MDA-MB-468, MDA-MB-231 or MCF-7 breast cancer cells were measured. The immunoreactivity of imported tat-mIgG was evaluated by measuring binding of ¹²³I--mFab to cell lysate and by displacement of binding of ¹²³I-mIgG to -mFab by cell lysate. Biodistribution and nuclear uptake of ¹²³I-tat-mIgG, ¹²³I-mIgG and ¹²³I-tat were compared in mice bearing s.c. MDA-MB-468 tumours.Results There was a 15-fold decrease in affinity of -mFab for tat-mIgG compared with mIgG. Internalized radioactivity imported into the nucleus for ¹²³I-tat-mIgG in MDA-MB-468, MDA-MB-231 and MCF-7 cells was 61.5±0.6%, 60.3±3.6% and 64.7±1.0%, respectively. The binding of ¹²³I--mFab to lysate from MDA-MB-468 cells importing tat-mIgG was 17-fold higher than that for cells not exposed to tat-mIgG. Imported tat-mIgG competed with tat-mIgG for displacement of binding of ¹²³I-mIgG to -mFab. Conjugation of mIgG to tat peptides did not change tissue distribution. Nuclear localization for ¹²³I-tat-mIgG in MDA-MB-468 tumours was 28.1±5.6%, and for liver, spleen and kidneys it was 41.7±2.7%, 13.8±0.8% and 36.9±3.3%, respectively.Conclusion ¹²³I-tat-mIgG radioimunoconjugates suggest a route to the design of radiopharmaceuticals exploiting intracellular and nuclear epitopes.     

冷束缚应激状态下大鼠内源性糖皮质激素与肠屏障功能损害的关系

目的观察冷束缚应激(cold restrain stress,CRS)状态下大鼠血清皮质醇浓度及肠屏障功能的改变情况,以及糖皮质激素受体阻断剂(RU38486)对CRS状态下大鼠肠屏障功能的影响。方法60只成年雄性SD大鼠,随机分为正常对照组、应激组(S组)和糖皮质激素受体阻断剂组(R组)。S组和R组按应激后检测时间又分为S2 h、S4 h、S8 h、S16 h、S24 h组和R2 h、R4 h、R8 h、R16 h、R24 h组。应激动物模型参照Brodie[1]的方法改良制作。动态观察各组大鼠的血皮质醇浓度变化及肠黏膜屏障功能的改变情况。结果应激组各时段大鼠血清皮质醇浓度、D-乳酸浓度、血浆内毒素浓度及肠黏膜损伤指数均高于正常组,而糖皮质激素受体阻断后血清皮质醇浓度、D-乳酸浓度、血浆内毒素浓度及肠黏膜损伤指数均较应激组相应各时段明显增高。结论应激可以造成肠黏膜上皮的损伤,导致肠黏膜屏障通透性增高,糖皮质激素受体阻断剂则进一步加重肠屏障功能的损害,表明应激产生的血清皮质醇对肠屏障功能起着保护性的作用。     

高海拔地区应用克氏针髓内固定治疗儿童尺桡骨干双骨折的临床疗效

目的：探讨克氏针髓内固定治疗高海拔地区儿童前臂双骨折的临床疗效。方法：2020年8月至2021年12月采用克氏针髓内固定治疗19例儿童尺桡骨干双骨折患者,男11例,女8例;年龄4~13（8.16±2.71）岁;病程1~10（4.11±2.51） d。首先尝试闭合复位,复位不成功则采取有限切开复位,再进行克氏针髓内固定尺桡骨。根据X线片评估骨折愈合情况,采用Anderson前臂功能评分标准评价疗效。结果：术后患者伤口愈合良好,其中术后发生针尾激惹临床表现2例,取出克氏针后症状消失。19例患者获随随访,时间3~14（7.68±3.50）个月。术后X线片示所有病例愈合,末次随访Anderson前臂功能评价：优16例,良2例,可1例。结论：高原地区儿童骨折往往就诊延误、医疗条件匮乏并且依从性不足,基于这些特点选用克氏针髓内固定治疗儿童尺桡骨双骨折具有损伤小,恢复快等优点,是一种可以推广的手术方式。