Surface-enhanced Raman scattering-based identification of breast cancer progression using extracellular vesicles-derived integrin α6ß4.

Detection of progression is of great importance to breast cancer treatment and can benefit patients. Limited by current detection technologies and biomarkers, early breast cancer progression diagnosis remains challenging. Researchers have found blood extracellular vesicles (EVs)-derived integrin α6ß4 directly facilitate progression in breast cancer, enabling cancer detection. However, EVs size and heterogeneity hinder protein detection, masked by abundant background EVs. Hence, novel tools for efficient detection of EVs with high selectivity and low interference are significantly desired. Here, a new silver-coated gold nanorods SERS probe, termed as Au@Ag@IDA-B/4MSTP, based on DNA aptamer was established for the detection of integrin α6ß4 derived from EVs. Validation of the Au@Ag@IDA-B/4MSTP probes using cell-culture-derived EVs revealed a LOD of 23 particles/µL for EVs detection. This tool was further confirmed to mimic the real state of cancer with subcutaneous tumor model and lung metastasis model in mice. With 10 µL of blood plasma and simple Raman analysis process, the test achieved 85.7 % sensitivity and 83.3 % specificity. Moreover, our method achieves a simplified approach that expedites the detection process. These results demonstrate the good detection performance of Au@Ag@IDA-B/4MSTP probes for EVs integrin α6ß4, and suggest that this non-invasive approach could be a promising tool for early detection of breast cancer progression.

Programing Immunogenic Cell Death in Breast Tumors with Designer DNA Frameworks.

The low response rate and serious side effects of cancer treatment pose significant limitations in immunotherapy. Here, we developed a multifunctional tetrahedral DNA framework (TDF) as a drug carrier to recruit chemotherapeutants and trigger immunogenic cell death (ICD) effects, which could turn tumors from cold to hot to boost the efficacy of antitumor immunotherapy. A tumor-targeting peptide RGD was modified on the TDF to increase the delivery efficiency, and the chemotherapeutant doxorubicin (DOX) was loaded to induce ICD effects, which were assisted by the immune adjuvant of CpG immunologic sequences linked on TDF. We demonstrated that the multifunctional TDF could suppress 4T1 breast tumor growth by increasing tumor infiltration of CD8+ T cells, upregulating granzyme B and perforin expressions to twice as much as the control group, and decreasing 30% CD25+ Treg cells. Furthermore, the combination of α-PD-1 could inhibit the growth of distant tumor and suppressed tumor recurrence in a bilateral syngeneic 4T1 mouse model; the distant tumor weight inhibition rate was about 91.6%. Hence, through quantitatively targeting the delivery of DOX to reduce the side effects of chemotherapy and sensitizing the immune response by ICD effects, this multifunctional TDF therapeutic strategy displayed better treatment effect and a promising clinical application prospect.

Proteomic Exploration of Endocytosis of Framework Nucleic Acids.

Efficient cell internalization of framework nucleic acid nanostructures free of transfection agents provides new opportunities for developing biocompatible and intelligent nanoprobes and drug delivery carriers. Here, a proteomic identification method to screen target proteins that interact with tetrahedral DNA nanostructures (TDNs) during the process of endocytosis by combining drug affinity responsive target stability (DARTS) with liquid chromatography/tandem mass spectrometry (LC-MS/MS) techniques, is reported. It is found that that caveolin-1 (CAV1) and macropinocytosis-related protein sorting nexin5 (SNX5) are associated with the endocytosis of TNDs, which is further validated by microscale thermophoresis (MST) analysis. CAV1- and SNX5- knockout experiments reveal that both caveolae-mediated endocytosis and macropinocytosis mediate the cellular uptake of TDNs, which complement previous findings with fluorescence tracing methods. This method provides a generic strategy to analyze cellular internalization process of DNA nanostructures for biomedical applications.

Hygroscopic properties of NaCl nanoparticles on the surface: a scanning force microscopy study.

We investigated the hygroscopic growth of sodium chloride (NaCl) nanoparticles with curvature related diameters ranging from 10 nm to 200 nm, at different relative humidities using scanning force microscopy. Hygroscopic aerosol nanoparticles play a vital role in the Earth's climate and human health. We report that 10 nm NaCl nanoparticles adsorbed on silicon surfaces have a higher deliquescence relative humidity than larger NaCl nanoparticles (size > 30 nm). This finding is consistent with the observations for airborne nanoparticles by hygroscopicity tandem differential mobility analyzer. Therefore, the presence of silicon surfaces plays no significant role in the deliquescence relative humidity. Moreover, the study of individual airborne particles by means of scanning force microscopy revealed that the ability of water uptake, i.e. growth factor, of NaCl particles differs by as large as 40% at the same relative humidity. This finding indicates that the individual nature of NaCl particles influences the growth factor.

Unbiased enrichment of urine exfoliated cells on nanostructured substrates for sensitive detection of urothelial tumor cells.

BACKGROUND: Early detection of urothelial carcinoma (UC) by noninvasive diagnostic methods with high accuracy is still underscored. This study aimed to develop a noninvasive assay incorporating both enrichment of urine exfoliated cells and immunoassays for UC detection. METHODS: Polystyrene dishes were exposed to oxygen plasma and modified with 3-aminopropyltriethoxysilane to prepare amine-functionalized nanostructured substrates (NS). Performance characterization of NS was evaluated by atomic force microscope and X-ray photoelectron spectroscopy. Urine exfoliated cells were captured by NS and then immunostained to detect urinary tumor cells (UTCs), which was called UTC assay. The receiver operating characteristic (ROC) curve, area under ROC curve (AUC), and Youden index were used to find the cutoff value of UTC assay. ROC analysis and McNemar test were used to compare the diagnostic accuracy of UTC assay with cytology. Kappa test was used to analyze the agreement of UTC assay and cytology with pathological diagnosis. RESULTS: Nanostructured substrates had good cell binding yields of nucleated cells and tumor cells. CK20+ CD45- CD11b- cells were considered as UTCs. UTC number ≥ 1 per sample could be considered as a positive result. By AUC and Kappa analysis, UTC assay showed good performance in UC detection. McNemar test demonstrated that UTC assay had a superior sensitivity even in low-grade subgroup and a similar specificity compared to cytology in UC diagnosis. CONCLUSIONS: Nanostructured substrates could be used to enrich the exfoliated cells from urine samples. UTC assay with NS has the potential to play a role in UC detection. The value of this assay still needs additional validation by large, multi-center studies.

Isolation and Retrieval of Extracellular Vesicles for Liquid Biopsy of Malignant Ground-Glass Opacity.

Extracellular vesicles (EVs) are cell-released vesicles of submicrometer size. EVs contain a tissue-specific signature wherein a variety of proteins and nucleic acids are selectively packaged. Recent studies validate that EVs can be used for cancer diagnostics, staging, and treatment monitoring. EV-related clinical translation requires effective EV isolation as a prerequisite. However, lengthy procedures, low yield, low throughput, and high levels of contaminants disqualify the existing isolation approaches for large-scale clinical use. Hence, new approaches for rapid, efficient, and low-cost isolation of EVs in high purity for flexible analyses of the diverse contents in real-world clinical settings are highly desired yet are currently unavailable. Here, we report the effective use of heparin/polymer-coated microspheres (HPM) for EV isolation and retrieval. Approximately 81% of EVs can be isolated from plasma in 1 h with depletion of ∼99.5% plasma protein and nucleic acid contaminants, and 72% of isolated EVs can be retrieved with saline in 5 min for various cargo analyses. This approach was further validated with clinical samples derived from patients with malignant ground-glass opacity (GGO). In eight patients, the mutation concordance between EV DNA and tissue DNA is 39.8%. The prevalence and mutation count of EGFR, TP53, and NF1 are higher than those of other oncogenes and antioncogenes that are intensely associated with lung adenocarcinoma. Moreover, different mutation prevalence and patterns between smokers and nonsmokers can be observed. Our findings suggest that the combination of HPM assay and targeted sequencing of EV DNA could be translated in the differential diagnosis of malignant GGO with short turnaround time.

Targeted Imaging of Brain Tumors with a Framework Nucleic Acid Probe.

Development of agents for delivering drugs and imaging probes across the blood-brain barrier (BBB) remains a major challenge. In this study, we designed a biocompatible framework nucleic acid (FNA)-based imaging probe for brain tumor-targeting. We employed a typical type of FNAs, tetrahedral DNA nanostructures (TDNs), as the building block, which were modified with angiopep-2 (ANG), a 19-mer peptide derived from human Kunitz domain of aprotinin. This probe exhibited high binding efficiency with low-density lipoprotein receptor-related protein-1 (LRP-1) of BBB and glioma. We found that ANG-functionalized TDNs (ANG-TDNs) stayed intact for at least 12 h in serum, and that ANG modification effectively enhanced cellular uptake of TDNs in brain capillary endothelial cells and Uppsala 87 malignant glioma (U87MG) cells. Remarkably, studies in both in vitro and in vivo models revealed that ANG-TDNs could cross the BBB. Especially, in vivo imaging showed strong fluorescent signals in U87MG human glioblastoma xenograft in nude mice. This study establishes that the FNA-based platform provides a new theranostic tool for the study and therapy of brain tumors.

Enzymatic O-GlcNAcylation of α-synuclein reduces aggregation and increases SDS-resistant soluble oligomers.

Neurodegenerative diseases including dementia with Lewy bodies, Lewy body variant of Alzheimer's disease, and Parkinson's disease are associated with the aberrant aggregation of α-synuclein, which is influenced by several post-translational modifications (PTMs). O-GlcNAcylation is one PTM that has an important role in many fundamental processes. The O-GlcNAcylation of endogenous α-synuclein at residues 53, 64, 72 and 87 has been reported in an unbiased mass spectrum analysis. The consequences of O-GlcNAcylation at residues 72 or 87 have been studied by using a synthetic α-synuclein bearing O-GlcNAcylation at threonine residue 72 or serine 87, respectively. O-GlcNAcylation at Thr72 or Ser87 suppresses the aggregation of α-synuclein. However, the effect of enzymatic O-GlcNAcylation of α-synuclein at multiple residues is not clear. Here, we successfully generated O-GlcNAcylated α-synuclein by co-expressing a shorter form of OGT (sOGT) with α-synuclein. The O-GlcNAcylation inhibited α-synuclein aggregation and promoted the formation of soluble SDS-resistant and Thioflavine T negative oligomers. Our data warrant further studies on the role of O-GlcNAcylation in the progression/treatment of Parkinson's disease in animal models.

Strong hydrophobic interaction between graphene oxide and supported lipid bilayers revealed by AFM.

Understanding the interaction between graphene oxide (GO) and lipid membranes is of great importance for its various applications in biotechnology. Here, we investigated the interaction between GO and charged supported lipid bilayers (SLBs) by in situ atomic force microscope (AFM) imaging. It was found that GO could peel off a single layer of positively charged SLBs and deposited on the hydrophobic part of the remaining sublayer. Then free lipid molecules would assemble on GO surface and formed 1.5 bilayers in a lipid-GO-lipid manner. For negatively charged lipid bilayers, however, GO deposited to the SLBs only when its concentration was very high. These results indicate that, in addition to electrostatic interaction, the hydrophobic interaction plays an important role when GO sheets deposit onto the charged lipid bilayers, and should be helpful to understand possible cytotoxicity and antibiosis of graphene-related nanomaterials. Microsc. Res. Tech. 79:721-726, 2016. © 2016 Wiley Periodicals, Inc.

Mechanical force-induced polymerization and depolymerization of F-actin at water/solid interfaces.

Actin molecules are among the three main cytoskeleton proteins of cells and undergo rapid cycling to regulate critical processes such as endocytosis, cytokinesis, cell polarity, and cell morphogenesis. Although extensive studies have been carried out on the dynamics as well as biological functions of actin polymerization and depolymerization both in vivo and in vitro, the molecular mechanisms by which cells sense and respond to mechanical signals are not fully understood. In particular, little attention has been paid to the effect of a physical force that is exerted directly on the actin cytoskeleton. In this paper, we have explored how the mechanical force affects the actin polymerization and depolymerization behaviors at water/solid interfaces using an atomic force microscope (AFM) operated in liquid. By raster scanning an AFM probe on a substrate surface with a certain load, it was found that actin monomers could polymerize into filaments without the help of actin related proteins (ARPs). Further study indicated that actin monomers were inclined to form filaments only under a small scanning load. The polymerized actin filaments would be depolymerized when the mechanical force was stronger. A possible mechanism has been suggested to explain the mechanical force induced actin polymerization.

Morphology change and detachment of lipid bilayers from the mica substrate driven by graphene oxide sheets.

Understanding the interaction between graphene oxide (GO) and a lipid membrane is significant for exploring the biocompatibility and cytotoxicity of GO, which is the basis for utilizing GO in the fields of biosensors, bioimaging, drug delivery, antibacterials, and so on. In this article, we monitored the dynamic process of the morphology change and detachment of lipid bilayers on mica substrates prompted by GO sheets by in situ atomic force microscope (AFM) imaging. It was found that the bare lipid bilayer dramatically expanded in height and would be unstable and detachable from the mica substrates as induced by GO. The detached lipid molecules were found to bind to the GO surface. The results also imply that GO is likely to influence the height and stability of the supported lipid bilayers (SLBs) by adsorbing metal ions such as calcium ions that were used to stabilize the bilayer structures on the mica substrate. These findings illustrate a complicated effect of GO on the SLBs and should be helpful in future applications of GO in biotechnology.

Salts drive controllable multilayered upright assembly of amyloid-like peptides at mica/water interface.

Surface-assisted self-assembly of amyloid-like peptides has received considerable interest in both amyloidosis research and nanotechnology in recent years. Despite extensive studies, some controlling factors, such as salts, are still not well understood, even though it is known that some salts can promote peptide self-assemblies through the so-called "salting-out" effect. However, they are usually noncontrollable, disordered, amorphous aggregates. Here, we show via a combined experimental and theoretical approach that a conserved consensus peptide NH2-VGGAVVAGV-CONH2 (GAV-9) (from representative amyloidogenic proteins) can self-assemble into highly ordered, multilayered nanofilaments, with surprising all-upright conformations, under high-salt concentrations. Our atomic force microscopy images also demonstrate that the vertical stacking of multiple layers is highly controllable by tuning the ionic strength, such as from 0 mM (monolayer) to 100 mM (mainly double layer), and to 250 mM MgCl2 (double, triple, quadruple, and quintuple layers). Our atomistic molecular dynamics simulations then reveal that these individual layers have very different internal nanostructures, with parallel ß-sheets in the first monolayer but antiparallel ß-sheets in the subsequent upper layers due to their different microenvironment. Further studies show that the growth of multilayered, all-upright nanostructures is a common phenomenon for GAV-9 at the mica/water interface, under a variety of salt types and a wide range of salt concentrations.

Adsorption of double-stranded DNA to graphene oxide preventing enzymatic digestion.

Investigation into the interactions between graphene oxide (GO) and biomolecules is very important for broad applications of GO in bioassay and bioanalysis. In this work, we describe the interactions between double-stranded DNA (dsDNA) and GO. We demonstrated that dsDNA can bind to GO forming complexes (dsDNA/GO) in the presence of certain salts, which protects dsDNA from being enzymatically digested. On the other hand, we found that a nonionic surfactant, such as triton X-100, can block the formation of dsDNA/GO complexes, so that the enzymatic digestion of dsDNA is restored. These results lead us to believe that the reason for GO protecting dsDNA from enzymatic digestion is the formation of dsDNA/GO complexes hindering the access of DNA enzymes to dsDNA, rather than direct inactivation of the DNA enzymes.