Atmosphere-inspired multilayered nanoarmor with modulable protection and delivery of Interleukin-4 for inflammatory microenvironment modulation.

Inflammatory bowel disease (IBD) has been closely associated with immune disorders and excessive M1 macrophage activation, which can be reversed by the M2-polarizing effect of interleukin-4 (IL-4). However, maintaining native IL-4 activity with its specific release in the inflammatory microenvironment and efficient biological performance remain a challenge. Inspired by the multilayered defense mechanism of the earth's atmosphere, we constructed a multilayered protective nanoarmor (NA) for IL-4 delivery (termed as IL-4@PEGRA NAs) into an intricate inflammatory microenvironment. The poly(ethylene glycol) (PEG)-ylated phenolic rosmarinic acid (RA)-grafted copolymer contains two protective layers-the intermediate polyphenol (RA molecules) and outermost shield (PEG) layers-to protect the biological activity of IL-4 and prolong its circulation in blood. Moreover, IL-4@PEGRA NAs scavenge reactive oxygen species with the specific release of IL-4 and maximize its biofunction at the site of inflammation, leading to M2 macrophage polarization and downregulation of inflammatory mediators. Simultaneously, gut microbiota dysbiosis can improve to amplify the M2-polarizing effect and inhibit the phosphatidylinositol 3 kinase/Akt signaling pathway, thereby attenuating inflammation and promoting colitis tissue repair. It provides a nature-inspired strategy for constructing an advanced multilayered NA delivery system with protective characteristics and potential for IBD management.

Composite nanofibrous dressing loaded with Prussian blue and heparin for anti-inflammation therapy and diabetic wound healing.

Diabetic ulcer is a severe complication of diabetes that can lead to amputation due to the overproduction of pro-inflammatory factors and reactive oxygen species (ROS). In this study, a composite nanofibrous dressing was developed by combining Prussian blue nanocrystals (PBNCs) and heparin sodium (Hep) through electrospinning, electrospraying, and chemical deposition. The nanofibrous dressing (PPBDH) was designed to take advantage of the excellent pro-inflammatory factor-adsorbing capability of Hep and the ROS-scavenging capabilities of PBNCs, resulting in synergistic treatment. It is worth noting that the nanozymes were firmly anchored to the fiber surfaces through slight polymer swelling caused by the solvent during electrospinning, thereby guaranteeing the preservation of the enzyme-like activity levels of PBNCs. The PPBDH dressing was found to be effective in reducing intracellular ROS levels, protecting cells from ROS-induced apoptosis, and capturing excessive pro-inflammatory factors, including chemoattractant protein-1 (MCP-1) and interleukin-1ß (IL-1ß). Furthermore, a chronic wound healing evaluation conducted in vivo demonstrated that the PPBDH dressing was able to effectively alleviate the inflammatory response and accelerate wound healing. This research presents an innovative approach to fabricate nanozyme hybrid nanofibrous dressings, which have great potential in accelerating the healing of chronic and refractory wounds with uncontrolled inflammation.

Sensitive Activatable Nanoprobes for Real-Time Ratiometric Magnetic Resonance Imaging of Reactive Oxygen Species and Ameliorating Inflammation In Vivo.

Imaging-guided real-time monitoring of the treatment process of inflammatory diseases is important for the timely adjustment of treatment planning to lower unnecessary side effects and improve treatment outcomes. However, it is difficult to reflect the dynamic changes of inflammation in vivo with enough tissue penetration depth. Here a novel nanotheranostic agent (denominated TMSN@PM) with platelet membrane (PM)-coated, tempol-grafted, manganese-doped, mesoporous silica nanoparticles is developed. The PM endows the TMSN@PM with the ability to target inflammation sites, which are verified by fluorescence imaging with Cyanine5 carboxylic acid (Cy5)-labeled TMSN@PM. Under the inflammatory environment (mild acidity and excess reactive oxygen species (ROS)), TMSN@PM can scavenge the excess ROS, thereby alleviating inflammation, degrade, and release manganese ions for enhanced magnetic resonance imaging (MRI). The relaxation changes (ΔR1 ) are almost linearly correlated with the concentration of H2 O2 , which can reflect the degree of inflammation. This method offers a non-invasive imaging-based strategy for early prediction of the therapeutic outcomes in inflammatory therapy, which may contribute to precision medicine in terms of prognostic stratification and therapeutic planning in future.

Exosomal circRNA HIPK3 knockdown inhibited cell proliferation and metastasis in prostate cancer by regulating miR-212/BMI-1 pathway.

Prostate cancer (PCa) is the second frequent malignancy among men in the world. Exosomal circular RNAs (circRNAs) have been reported to function in PCa progression. The current study aimed to investigate the role of exosomal circRNA homeodomain-interacting protein kinase 3 (circHIPK3) in PCa development. Exosomes were extracted from serum and cells utilizing commercial kit, and identified by transmission electron microscopy (TEM), Western blot assay and nanoparticle tracking analyzer. Relative expression of circHIPK3, microRNA (miR)-212 and B-cell specific MMLV insertion site-1 (BMI-1) was examined by quantitative realtime PCR or Western blot assay. Receiver Operating Characteristic (ROC) analysis was conducted to assess the diagnostic potential of exosomal miR-212. Cell viability, and metastasis including migration and invasion, were detected by Methyl thiazolyl tetrazolium (MTT) assay and Transwell assay, respectively. Cell apoptosis was monitored using flow cytometry. The interaction between miR-212 and circHIPK3 or BMI-1 was validated by dual-luciferase reporter assay. Xenograft tumor assay was employed to explore the role of exosomal circHIPK3 in vivo. Exosomal circHIPK3 was increased in serum of PCa patients, and could discriminate PCa patients from normal volunteers. Depletion of exosomal circHIPK3 or overexpression of exosomal miR-212 reduced viability, migration and invasion, but promoted cell apoptosis in PCa cells, which was attenuated by miR-212 inhibition or BMI-1, respectively. MiR-212 targeted BMI-1, and downregulated BMI-1 expression. Exosomal circHIPK3 knockdown also suppressed tumor growth in vivo. Exosomal circHIPK3 knockdown inhibited PCa progression by regulating miR-212/BMI-1 axis, at least in part, offering a new insight into the molecular mechanism of PCa.

Evolution of DNA aptamers through in vitro metastatic-cell-based systematic evolution of ligands by exponential enrichment for metastatic cancer recognition and imaging.

Metastasis, the capability of tumor cells to spread and grow at distant sites, is the primary factor in cancer mortality. Because metastasis in sentinel lymph nodes suggests the original spread of tumors from a primary site, the detection of lymph node involvement with cancer serves as an important prognostic and treatment parameter. Here we have developed a panel of DNA aptamers specifically binding to colon cancer cell SW620 derived from metastatic site lymph node, with high affinity after 14 rounds of selection by the cell-SELEX (systematic evolution of ligands by exponential enrichment) method. The binding affinities of selected aptamers were evaluated by flow cytometry. Aptamer XL-33 with the best binding affinity (0.7 nM) and its truncated sequence XL-33-1 with 45 nt showed excellent selectivity for recognizing target cell SW620. The binding entity of the selected aptamer has been preliminarily determined as a membrane protein on the cell surface. Tissue imaging results showed that XL-33-1 was highly specific to the metastatic tumor tissue or lymph node tissue with corresponding cancer metastasis and displayed an 81.7% detection rate against colon cancer tissue with metastasis in regional lymph nodes. These results suggest that XL-33-1 has great potential to become a molecular imaging agent for early detection of lymph node tissue with colon cancer metastasis. More importantly, this study clearly demonstrates that DNA ligands selectively recognizing metastatic cancer cells can be readily generated by metastatic-cell-based SELEX for potential applications in metastatic cancer diagnosis and treatment.

Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity.

Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher's attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with Kd 56±7.3nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

A multifunctional nanomicelle for real-time targeted imaging and precise near-infrared cancer therapy.

Simultaneous targeted cancer imaging, therapy and real-time therapeutic monitoring can prevent over- or undertreatment. This work describes the design of a multifunctional nanomicelle for recognition and precise near-infrared (NIR) cancer therapy. The nanomicelle encapsulates a new pH-activatable fluorescent probe and a robust NIR photosensitizer, R16FP, and is functionalized with a newly screened cancer-specific aptamer for targeting viable cancer cells. The fluorescent probe can light up the lysosomes for real-time imaging. Upon NIR irradiation, R16FP-mediated generation of reactive oxygen species causes lysosomal destruction and subsequently trigger lysosomal cell death. Meanwhile the fluorescent probe can reflect the cellular status and in situ visualize the treatment process. This protocol can provide molecular information for precise therapy and therapeutic monitoring.

In vitro selection of DNA aptamers for metastatic breast cancer cell recognition and tissue imaging.

Cancer is a major public health issue, with metastatic cancer accounting for the overwhelming majority of cancer deaths. Early diagnosis and appropriate treatment of metastatic cancer may largely prolong the survival rate and improve the quality of life for patients. In this study, we have identified a panel of DNA aptamers specifically binding to MDA-MB-231 cells derived from metastatic site-pleural effusion, with high affinity after 15 rounds of selections using the cell-based systematic evolution of ligands by exponential enrichment (SELEX) method. The selected aptamers were subjected to flow cytometry and laser confocal fluorescence microscopy to evaluate their binding affinity and selectivity. The aptamer LXL-1 with the highest abundance in the enriched library demonstrated a low K(d) value and excellent selectivity for the recognition of the metastatic breast cancer cells. Tissue imaging results showed that truncated aptamer sequence LXL-1-A was highly specific to the corresponding tumor tissue and displayed 76% detection rate against breast cancer tissue with metastasis in regional lymph nodes. Therefore, on the basis of its excellent targeting properties and functional versatility, LXL-1-A holds great potential to be used as a molecular imaging probe for the detection of breast cancer metastasis. Our result clearly demonstrates that metastatic-cell-based SELEX can be used to generate DNA ligands specifically recognizing metastatic cancer cells, which is of great significance for metastatic cancer diagnosis and treatment.

Identification, characterization and application of a G-quadruplex structured DNA aptamer against cancer biomarker protein anterior gradient homolog 2.

BACKGROUND: Anterior gradient homolog 2 (AGR2) is a functional protein with critical roles in a diverse range of biological systems, including vertebrate tissue development, inflammatory tissue injury responses, and cancer progression. Clinical studies have shown that the AGR2 protein is overexpressed in a wide range of human cancers, including carcinomas of the esophagus, pancreas, breast, prostate, and lung, making the protein as a potential cancer biomarker. However, the general biochemical functions of AGR2 in human cells remain undefined, and the signaling mechanisms that drive AGR2 to inhibit p53 are still not clearly illustrated. Therefore, it is of great interest to develop molecular probes specifically recognizing AGR2 for its detection and for the elucidation of AGR2-associated molecular mechanism. METHODOLOGY/PRINCIPAL FINDINGS: Through a bead-based and flow cytometry monitored SELEX technology, we have identified a group of DNA aptamers that can specifically bind to AGR2 with K(d) values in the nanomolar range after 14 rounds of selections. Aptamer C14B was chosen to further study, due to its high binding affinity and specificity. The optimized and shortened C14B1 has special G-rich characteristics, and the G-rich region of this binding motif was further characterized to reveal an intramolecular parallel G-quadruplex by CD spectroscopy and UV spectroscopy. Our experiments confirmed that the stability of the G-quadruplex structure was strongly dependent on the nature of the monovalent ions and the formation of G-quadruplex structure was also important for the binding capacity of C14B1 to the target. Furthermore, we have designed a kind of allosteric molecule beacon (aMB) probe for selective and sensitive detection of AGR2. CONCLUSION/SIGNIFICANCE: In this work, we have developed new aptamer probes for specific recognition of the AGR2. Structural study have identified that the binding motif of aptamer is an intramolecular parallel G-quadruplex structure and its structure and binding affinity are strongly dependent on the nature of the monovalent ion. Furthermore, with our design of AGR2-aMB, AGR2 could be sensitively and selectively detected. This aptamer probe has great potential to serve as a useful tool for early diagnosis and prognosis of cancer and for fundamental research to elucidate the biochemical functions of AGR2.

Selection of DNA aptamers against glioblastoma cells with high affinity and specificity.

BACKGROUND: Glioblastoma is the most common and most lethal form of brain tumor in human. Unfortunately, there is still no effective therapy to this fatal disease and the median survival is generally less than one year from the time of diagnosis. Discovery of ligands that can bind specifically to this type of tumor cells will be of great significance to develop early molecular imaging, targeted delivery and guided surgery methods to battle this type of brain tumor. METHODOLOGY/PRINCIPAL FINDINGS: We discovered two target-specific aptamers named GBM128 and GBM131 against cultured human glioblastoma cell line U118-MG after 30 rounds selection by a method called cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX). These two aptamers have high affinity and specificity against target glioblastoma cells. They neither recognize normal astraglial cells, nor do they recognize other normal and cancer cell lines tested. Clinical tissues were also tested and the results showed that these two aptamers can bind to different clinical glioma tissues but not normal brain tissues. More importantly, binding affinity and selectivity of these two aptamers were retained in complicated biological environment. CONCLUSION/SIGNIFICANCE: The selected aptamers could be used to identify specific glioblastoma biomarkers. Methods of molecular imaging, targeted drug delivery, ligand guided surgery can be further developed based on these ligands for early detection, targeted therapy, and guided surgery of glioblastoma leading to effective treatment of glioblastoma.