The Effects of the Carrier and Ligand Spatial Conformation on RNA Nanodrug Cell Delivery.

Small interfering RNA (siRNA) highlights the immense therapeutic potential for cancer treatment. The major challenge in siRNA therapy is the effective RNA nanodrug delivery system, which is facilitated by the ligand and the carrier. In this study, we analyzed the binding specificity of linear RGD and circular RGD to αVß3 integrins by mapping the morphology using super-resolution direct stochastic optical reconstruction microscopy. Meanwhile, the binding dynamics was investigated using single-molecule force spectroscopy. Then, the effects of the ligand and carrier on RNA nanodrug cell entry dynamic parameters were evaluated at the single particle level by the force tracing technique. Furthermore, the delivery efficiency of RNA nanodrugs was assessed using AFM-based nanoindentation at the single cell level. This report will provide valuable insights for rational design strategies aiming to achieve improved efficiency for nanodrug delivery systems.

Revealing the dynamic mechanism of cell-penetrating peptides across cell membranes at the single-molecule level.

Cell-penetrating peptides (CPPs) have gained prominence in cellular drug delivery due to their extremely low toxicity and rapid cell internalization properties. Understanding the effect of CPPs' physicochemical properties on trans-membrane behavior will provide a better screening scheme for designing effective CPP-conjugated nano-drugs. Herein, the efficiency of the CPPs interacting with the cell membrane and the subsequent trans-membrane is revealed at the single-molecule level using single-molecule force spectroscopy (SMFS) and force tracing technique based on atomic force spectroscopy (AFM). The dynamic force spectroscopy (DFS) analysis indicates that cationic TAT48-60 and amphipathic MAP are more effective during the interaction with cell membrane due to the strong electrostatic interaction between CPPs and cell membrane. However, for the subsequent trans-membrane process, the hydrophobicity of Pep-7 plays a key role, showing a higher trans-membrane speed at the single-molecule level. Meanwhile, Pep-7 shows lower trans-membrane speed and probability on normal cells (Vero), which makes it more suitable as a peptide-based nano-drug to treat tumors avoiding harming normal cells. The dynamic parameters obtained in this study offer guidance for screening and modifying effective CPPs, targeting specific cell lines or tissues during the nano-drug design.

Exploring the role of exosomal MicroRNAs as potential biomarkers in preeclampsia.

The complex pathogenesis of preeclampsia (PE), a significant contributor to maternal and neonatal mortality globally, is poorly understood despite substantial research. This review explores the involvement of exosomal microRNAs (exomiRs) in PE, focusing on their impact on the protein kinase B (AKT)/hypoxia-inducible factor 1-α (HIF1α)/vascular endothelial growth factor (VEGF) signaling pathway as well as endothelial cell proliferation and migration. Specifically, this article amalgamates existing evidence to reveal the pivotal role of exomiRs in regulating mesenchymal stem cell and trophoblast function, placental angiogenesis, the renin-angiotensin system, and nitric oxide production, which may contribute to PE etiology. This review emphasizes the limited knowledge regarding the role of exomiRs in PE while underscoring the potential of exomiRs as non-invasive biomarkers for PE diagnosis, prediction, and treatment. Further, it provides valuable insights into the mechanisms of PE, highlighting exomiRs as key players with clinical implications, warranting further exploration to enhance the current understanding and the development of novel therapeutic interventions.

Incidence, prognostic factors, and a nomogram of cervical cancer with lung metastasis: A SEER-based study.

AIMS: The purpose of this study was to investigate the incidence, survival and prognostic factors of cervical cancer with lung metastasis at the initial diagnosis and to develop a visual nomogram to predict the prognosis of these patients. METHODS: We used the Surveillance, Epidemiology and End Results (SEER) database to screen patients diagnosed with cervical cancer from 2010 to 2015. After strict inclusion and exclusion, the chi-square test was used to evaluate the differences in the clinical characteristics of patients with cervical cancer, and then we used Kaplan-Meier method to perform survival analysis among cervical cancer patients with lung metastasis. Next, univariate and multivariate Cox proportional hazard regression models were used to estimate prognostic factors of these patients and we developed a visualized and novel nomogram to judge the prognosis. RESULTS: 476 patients with lung metastasis and 12,016 patients without lung metastasis were included in this study. The incidence of lung metastasis was higher in unmarried white cervical cancer patients between the ages of 40 and 60, and grade III cervical squamous cell carcinoma patients were more likely to have lung metastasis. In addition, grade, surgery, radiotherapy, sequence of surgery and radiotherapy and chemotherapy were significantly related to the outcomes of cervical cancer patients with lung metastasis. Furthermore, our nomogram could predict the 3-year and 5-year overall survival (OS) of these patients. Finally, the AUC of 3-year OS and 5-year OS were confirmed to be 0.969 and 0.939 respectively by ROC curves, with good consistency. CONCLUSIONS: Age at diagnosis, race, marital status, and characteristics of the tumor can influence the incidence of lung metastasis in cervical cancer patients. Besides, grade, surgery, radiotherapy, sequence of surgery and radiotherapy and chemotherapy may deeply affect the prognosis of cervical cancer patients with lung metastasis. The nomogram built in this study may help clinicians to formulate individualized treatment strategies and encourage the development of more and more comprehensive and accurate predictive models.

Factors associated with human papillomavirus persistence after loop electrosurgical excision procedure in patients with cervical squamous intraepithelial lesion.

AIM: To seek the high-risk factors of human papillomavirus (HPV) persistence and residual lesion or recurrence after loop electrosurgical excision procedure (LEEP) focus on the predictive value of intraoperative human papilloma virus (IOP-HPV) testing. METHODS: Intraoperative endocervical sample was obtained with a cytobrush from the remained cervix of 292 patients immediately after LEEP. HPV Genotyping was performed using a polymerase chain reaction technique. All patients followed by HPV genotyping and cytology every 3-6 months. The IOP-HPV testing results and possible risk factors such as age, cytology grade, menopause status, margin involvement, preoperative HPV status, and cervical lesion grade were assessed in predicting persistence of HPV and residual lesion or recurrence after surgery. RESULTS: There were 61 (20.9%) patients presented persistent HPV infection. Multivariate analyses showed that IOP-HPV positive, post-menopause and preoperative HPV multiplex infection was strongly associated with HPV persistence after LEEP, IOP-HPV positive and post-menopause was also associated with residua or recurrent disease after LEEP. CONCLUSIONS: IOP-HPV positive, post-menopause, and preoperative HPV multiplex infection are independent predictors of HPV persistence in patients with cervical squamous intraepithelial lesion treated by LEEP. IOP-HPV test is a new approach that may potentially allow for early identification of patients at high risk of HPV persistence and residua or recurrent disease after LEEP, thereby possibly facilitate an attenuated follow-up schedule for negative patients those at low risk of persistent HPV infection.

Distinguishing the Cellular Transport of Folic Acid Conjugated Nano-Drugs among Different Cell Lines by Using Force Tracing Technique.

Folic acid (FA) is a ligand that has been renowned for its strong binding to FA receptor (FR), and the robustness of the specific interaction has led to the generation of multitudinous tumor-targeted nano-drug delivery systems. However, selecting the appropriate FA targeted nano-drugs according to types of cancerous cells to achieve a high effect is critical. Understanding of how the drug is transported through the cell membrane and is delivered intracellularly is very important in screening ideal targeted nano-drugs for cancerous changes in different organs. Herein, by using a force tracing technique based on atomic force microscopy (AFM), the dynamic process of FA-polyamidoamine-Doxorubicin (FA-PAMAM-DOX) entry into different tumor cells (HeLa and A549) and normal cells (Vero) was monitored in real time. The cell membrane transport efficacy of FA-PAMAM-DOX in tumor cells with an FR high overexpression level (HeLa) and FR low overexpression level (A549) is analyzed, which is significantly higher than that in normal cells (Vero), especially for HeLa cells. Subsequently, the intracellular delivery efficiency of FA-PAMAM-DOX in different cell lines was measured by using fluorescence imaging and AFM-based nanoindentation techniques. This report will help to discover the cellular transport mechanism of nano-drugs and screen out optimal therapeutic nano-drugs for different types of tumors.

Investigating the trans-membrane transport of HAIYPRH peptide-decorated nano-drugs.

Transferrin (Tf) has been effectively used to promote the cellular uptake of HAIYPRH (T7) peptide-conjugated nano-drugs. In this study, the enhancing effect of Tf on T7-decorated nano-drug transport was investigated using force tracing and nano-indentation techniques at a single-particle/cell level. Furthermore, the results were confirmed by ensemble fluorescence imaging.

Incidence, prognostic factors and a nomogram of cervical cancer with distant organ metastasis: a SEER-based study.

This study was to investigate the incidence, survival and prognostic factors of cervical cancer with distant organ metastasis, and to develop a nomogram to predict the prognosis of cervical cancer. We used the Surveillance, Epidemiology and End Results (SEER) database to screen patients diagnosed with cervical cancer from 2010 to 2014. The chi-squared test was used to analyse the differences in clinical characteristics, and we used Kaplan-Meier methods to perform survival analysis. Univariate and multivariate Cox proportional hazard regression models were used to estimate prognostic factors, and we developed a visual nomogram to judge the prognosis. We found that lung metastasis was the most common in cervical cancer patients with distant organ metastasis. Age, race, characteristics of the tumour, and therapy should be considered when analysing the prognosis of cervical cancer patients. The findings of this study may help clinicians to formulate individualised treatment strategies.Impact StatementWhat is already known on this subject? Distant organ metastasis of cervical cancer mainly involves lung, bone, liver and brain. Once it occurs, the survival and prognosis will be threatened seriously.What the results of this study add? 4176 patients were included, and lung metastasis was the most common in cervical cancer with distant organ metastasis (3.5%). Additionally, age, race, tumour grade, histological type, T-stage, N-stage, lung, liver and bone metastasis and the treatment mode are significantly related to the outcomes of cervical cancer patients. Furthermore, we developed a nomogram that could predict the probability of three-year and five-year OS.What the implications are of these findings for clinical practice and/or further research? The findings of this study may drive more and more studies focussing on the comprehensive prognostic assessment, diagnosis, and treatment of distant metastasis of cervical cancer. Besides, clinicians can utilise these findings to formulate individualised treatment strategies.

Evaluating the therapeutic efficacy of nano-drugs targeting epidermal growth factor receptor.

Epidermal growth factor receptor (EGFR) targeted nano-drugs facilitate effective diagnosis and treatment of cancer. Herein, the therapeutic efficacy of nano-drugs targeting EGFR was evaluated from the perspective of cell entry efficiency and induced cell mechanical properties using force tracing and nano-indentation techniques at the single particle/cell level in real time.

Evaluating the single-molecule interactions between targeted peptides and the receptors on living cell membrane.

As potential ligands, targeted peptides have become an important part in the construction of intelligent drug delivery systems (DDSs). The targeting interaction of peptides with receptors is a key point affecting the efficacy of targeted nano-drugs. Herein, three common peptides (HAIYPRH (T7), YHWYGYTPQNVI (GE11), and RGD) that have been widely used in cancer targeted therapy and tumor diagnostics, targeting the corresponding receptors (transferrin receptor (TfR), epidermal growth factor receptor (EGFR), and ανß3 integrin receptor), were selected as examples to study the targeting interacton on living cell surface at the single-molecule level by using single-molecule force spectroscopy (SMFS) based on atomic force microscopy (AFM). The dissociation activation energy in the absence of an external force (ΔGß,0) of T7-TfR, GE11-EGFR, and RGD-ανß3 integrin is evaluated at single-molecule level. Among these three peptide-receptor pairs, the T7-TfR bond is the most stable with a smaller dissociation kinetic rate constant at zero force (Koff), larger kinetic on-rate constant (Kon), and shorter interaction time (τ). Furthermore, T7 can target TfR even more effectively on A549 cell membrane after treatment with drugs. Our methodology can also be applicable to the study of other ligand targeted DDSs.

Revealing the Dynamic Mechanism by Which Transferrin Promotes the Cellular Uptake of HAIYPRH Peptide-Conjugated Nanostructures by Force Tracing.

The HAIYPRH (T7) peptide has been widely used as a ligand for constructing tumor-targeted nanodrug delivery systems since it can target the transferrin receptor (TfR) and then enter cells easily with the help of transferrin (Tf). However, the dynamic mechanism by which transferrin promotes the entry of T7-conjugated nanostructures into cells remains unclear. Herein, a force tracing technique based on atomic force microscopy (AFM) was used to track the ultrafast dynamic process of a T7-conjugated gold nanoparticle (AuNP-T7) entering a cell at the single-particle level in real time. Tf helped decrease the endocytosis force and increase the endocytosis speed of AuNP-T7 in A549 cells. However, Tf only increased the endocytosis speed of AuNP-T7 in HeLa cells. In contrast, in Vero cells without TfR overexpression, Tf decreased the endocytosis speed. This report provides important insights for redesigning and developing T7-conjugated nanodrug carriers in targeted nanodrug delivery systems.

Dynamics of delivering aptamer targeted nano-drugs into cells.

A targeted nano-drug delivery system has provided great potential and benefits to the diagnosis and therapy of cancers. Cell entry is a critical step for taking effect of the targeted nano-drug. In this report, the dynamics of delivering a single aptamer targeted polyamindoamine-camptothecin-AS1411 (PAMAM-CPT-AS1411) nano-drug into cells was investigated using a force tracing technique based on atomic force microscopy. The results show that the specific interaction of AS1411 and nucleolin, which is overexpressed on cancer cells, enhances the efficiency of the PAMAM-CPT-AS1411 cell entry. Moreover, the specific interaction induced receptor-mediated endocytosis prolongs the duration and decreases the speed of a single PAMAM-CPT-AS1411 cell entry, which is helpful to understand the targeted nano-drugs prolonging the therapeutic drug level. However, the required force for PAMAM-CPT-AS1411 cell entry is not changed. This report will provide a novel and potential method for achieving the precise dynamics of targeted nano-drug delivery.

Size-Dependent Transmembrane Transport of Gold Nanocages.

Gold nanocages (Au NCs), as drug carriers, have been widely applied for cancer diagnosis and photothermal therapy (PTT). Transmembrane transporting efficacy of Au NCs is the fundamental and important issue for their use in PTT. Herein, we used a force tracing technique based on atomic force microscopy to track the dynamic transmembrane process of Au NCs at the single-particle level in real time. Meanwhile, we measured and compared the dynamic parameters of Au NCs with sizes of 50 and 100 nm usually used as nanodrug carriers of PTT. It is concluded that the 50 nm Au NC transmembrane transporting needs smaller force and shorter duration with a much faster speed. However, both the 50 and 100 nm Au NC transmembrane transporting depends on the caveolin-mediated endocytosis, clathrin-mediated endocytosis, and macropinocytosis, which was also confirmed by confocal fluorescence imaging. This report will provide a potential technique for screening nanodrug carriers from the perspective of transmembrane transporting efficacy.

Tracking the Single-Carbon-Dot Transmembrane Transport by Force Tracing Based on Atomic Force Microscopy.

Carbon dots (CDs) evoke a great deal of attention in biomedicine because of their unique properties. As an emerging theranostic agent, the CDs transmembrane transport process is the first and the most important step. Herein, a transmembrane dynamic process of transporting single CDs with folic acid into normal cells (Vero) and cancer cells (HeLa) were tracked by means of a force tracing technique based on atomic force microscopy. Meanwhile, the kinetic parameters of the the transmembrane process were measured and calculated. Interestingly, in the comparison between cancer cells (HeLa) and normal cells (Vero), the transporting force of single CDs with folic acid was smaller and the transporting average speed was slower.

ROS-Mediated Cell Cycle Arrest and Apoptosis Induced by Zearalenone in Mouse Sertoli Cells via ER Stress and the ATP/AMPK Pathway.

Zearalenone (ZEA) can perturb the differentiation of cells, reduce the generation of reproductive cells and induce a death of germ cells, but the molecular mechanism remains unclear. In order to investigate the potential mechanism of ZEA-induced cell cycle arrest and apoptosis, we studied the effects of ZEA on cell proliferation, cell-cycle distribution, cell-cycle-related proteins, cell death, cell apoptosis, ROS generation and the ATP/AMPK pathway in Sertoli cells. The role of ROS, ER stress and the ATP/AMPK pathway in ZEA-induced cell-cycle arrest and cell apoptosis was explored by using the antioxidant NAC, ER stress inhibitor 4-PBA and the AMPK inhibitor dorsomorphin, respectively. The results revealed that ZEA inhibited the cell proliferation, influenced the distribution of the cell cycle and induced cell apoptosis through the ATP/AMPK pathway. The ATP/AMPK pathway was regulated by ER stress that was induced by ROS generation after exposure to ZEA. Taking these together, this study provided evidence that ROS regulated the process of ZEA-induced cell cycle arrest and cell apoptosis through ER stress and the ATP/AMPK signal ways.

Evaluating the efficacy of the anticancer drug cetuximab by atomic force microscopy.

Cetuximab is a monoclonal antibody that binds to the epidermal growth factor receptor, which is important in the growth of many cancers. However, the biophysical characteristics of cetuximab as an anti-cancer drug remain elusive. In this study, we adopted atomic force microscopy to measure the mechanical properties of cancer cells following cetuximab treatment and the biomechanical properties of cetuximab and epidermal growth factor receptor interactions. Atomic force microscopy can be implemented as a platform for further investigations that target the cellular stiffness and affinity of ligand-receptor as a therapeutic choice.

Specificity and mechanism of action of alpha-helical membrane-active peptides interacting with model and biological membranes by single-molecule force spectroscopy.

In this study, to systematically investigate the targeting specificity of membrane-active peptides on different types of cell membranes, we evaluated the effects of peptides on different large unilamellar vesicles mimicking prokaryotic, normal eukaryotic, and cancer cell membranes by single-molecule force spectroscopy and spectrum technology. We revealed that cationic membrane-active peptides can exclusively target negatively charged prokaryotic and cancer cell model membranes rather than normal eukaryotic cell model membranes. Using Acholeplasma laidlawii, 3T3-L1, and HeLa cells to represent prokaryotic cells, normal eukaryotic cells, and cancer cells in atomic force microscopy experiments, respectively, we further studied that the single-molecule targeting interaction between peptides and biological membranes. Antimicrobial and anticancer activities of peptides exhibited strong correlations with the interaction probability determined by single-molecule force spectroscopy, which illustrates strong correlations of peptide biological activities and peptide hydrophobicity and charge. Peptide specificity significantly depends on the lipid compositions of different cell membranes, which validates the de novo design of peptide therapeutics against bacteria and cancers.

Scanning Ion Conductance Microscopic Study for Cellular Uptake of Cationic Conjugated Polymer Nanoparticles.

Positively charged conjugated polymer nanoparticles (CPNs) are emerging biomaterials exhibiting high levels of cellular entry. High rate of cellular entry efficiency is believed that the amphiphilic CPNs interact efficiently with the negatively charged hydrophobic cellular membranes. For the first time, the cell surface morphological changes of human cervical cancer cells treated with CPNs using a scanning probe microscopy technique, scanning ion conductance microscopy (SICM) are imaged. After 1 h of CPN incubation, distinct changes are observed in cell surface morphology such as interconnected protrusions and pits with sub-micrometer sizes, which are not observed from cells treated with positively charged polyethyleneimine (PEI) under the same treatment conditions. The change on cell surface morphology is quantified by surface roughness ratio, which is increased as CPN concentration increases, while the ratio first increases and then decreases as the incubation time increases. These results suggest that cells respond actively toward CPN with both positive charges on the side chain and the hydrophobicity from rigid aromatic backbone, which leads to subsequent endocytosis. In conclusion, it is demonstrated that SICM is a suitable imaging technique to reveal the dynamic alternations on the cell surface morphology at the early stage of nanoparticles endocytosis with high resolution.

The force of transporting a single amino acid into the living cell measured using atomic force microscopy.

We used single molecule force spectroscopy (SMFS) to investigate the interacting force between single cysteine and amino acid transporters in eukaryotic cell membranes. We measured the transporting force of cysteine and found that its conformation on the AFM tip is important for discriminating the substrate in the transporting pathway.

The study of single anticancer peptides interacting with HeLa cell membranes by single molecule force spectroscopy.

To determine the effects of biophysical parameters (e.g. charge, hydrophobicity, helicity) of peptides on the mechanism of anticancer activity, we applied a single molecule technique-force spectroscopy based on atomic force microscope (AFM)-to study the interaction force at the single molecule level. The activity of the peptide and analogs against HeLa cells exhibited a strong correlation with the hydrophobicity of peptides. Our results indicated that the action mode between α-helical peptides and cancer cells was largely hydrophobicity-dependent.