Astrocyte-targeted siRNA delivery by adenosine-functionalized LNP in mouse TBI model.

Traumatic brain injury (TBI) induces pro-inflammatory polarization of astrocytes and causes secondary disruption of the blood-brain barrier (BBB) and brain damage. Herein, we report a successful astrocyte-targeted delivery of small interfering RNA (siRNA) by ligand functionalized lipid nanoparticles (LNPs) formulated from adenosine-conjugated lipids and a novel ionizable lipid (denoted by Ad4 LNPs). Systemic administration of Ad4 LNPs carrying siRNA against TLR4 to the mice TBI model resulted in the specific internalization of the LNPs by astrocytes in the vicinity of damaged brain tissue. A substantial knockdown of TLR4 at both mRNA and protein levels in the brain was observed, which led to a significant decrease of key pro-inflammatory cytokines and an increase of key anti-inflammatory cytokines in serum. Dye leakage measurement suggested that the Ad4-LNP-mediated knockdown of TLR4 attenuated the TBI-induced BBB disruption. Together, our data suggest that Ad4 LNP is a promising vehicle for astrocyte-specific delivery of nucleic acid therapeutics.

Colorimetric/photothermal dual-mode sensing detection of ascorbic acid based on a Ag[i] ion/3,3',5,5'-tetramethylbenzidine (TMB) system.

In this work, a novel strategy of colorimetric and photothermal dual-mode sensing determination of ascorbic acid (AA) based on a Ag+/3,3',5,5'-tetramethylbenzidine (TMB) system was developed. In this sensing system, Ag+ could oxidize TMB with a distinct color change from colorless to blue color, strong absorbance at 652 nm and a photothermal effect under 808 nm laser irradiation due to the formation of oxidized TMB (oxTMB). When AA was present, oxTMB was reduced accompanied by a change from blue to colorless, and a decrease in absorption peak intensity and the photothermal effect. AA concentration showed a negative linear correlation with the value of both the absorbance intensity at 652 nm and temperature in the range of 0.2-10 µM (A = -0.03C + 0.343 (R 2, 0.9887; LOD, 50 nM); ΔT = -0.57C + 8.453 (R 2, 0.997; LOD, 7.8 nM)). Based on this, a sensing approach for detection of AA was proposed with dual-mode and without the complicated synthesis of nanomaterials. The photothermal effect and colorimetric signal provided a dual-mode detection strategy for AA, overcoming the limitations of any single mode. This colorimetric and photothermal dual-mode detection has great potential in the detection of AA in clinical pharmaceuticals and the construction of portable and highly sensitive sensors.

Identification of a novel DNA aptamer that selectively targets lung cancer serum.

Lung cancer is the leading cause of cancer-related deaths worldwide. Early diagnosis and treatment is critical to improving the 5 year survival rate of lung cancer. The identification of new options for early-stage diagnosis and therapy of lung cancer still represents a crucial challenge. Therefore, a new diagnostic method is urgently needed. In this study, we used a new modified SELEX, called serum-SELEX, to isolate aptamers that can specifically bind lung cancer serum, without any prior knowledge of their target. Among the obtained candidate aptamer sequences, Ap-LC-19 was identified as the optimal aptamer probe with the lowest dissociation constant (K d) value of 15 ± 8.6 nM and higher affinity assessed by qPCR. Furthermore, this molecule could be a suitable aptamer for lung cancer serum and could be used as a recognition element in aptamer-based biosensors for efficient early diagnosis of lung cancer or as an innovative tool for targeted therapy. In addition, we performed MALDI-TOF MS followed by secondary peptide sequencing MS analysis for the identification of the aptamer targeted proteins. CLEC3B could be useful biomarkers for early detection of lung cancer and in monitoring its evolution.

In vitro selection of DNA aptamers for the development of chemiluminescence aptasensor for neuron-specific enolase (NSE) detection.

Neuron-specific enolase (NSE) is one of the most commonly used serum tumor biomarker in clinical practice for small cell lung cancer screening, early diagnosis, evaluation of therapeutic efficacy and prognosis. In this study, we obtained DNA aptamers with great affinity and selectivity to NSE via subtractive SELEX approach. After 10 rounds, three candidate aptamers were successfully selected and identified. Their affinities were measured by surface plasmon resonance. Apt-5 aptamer with high binding affinity and good specificity were obtained, which had the dissociation constant (K D) values of 12.26 nM. In addition, electrophoretic mobility shift assay (EMSA) experiment also further indicated that the Apt-5 had a highly specific affinity to NSE without binding to HSA. The circular dichroism (CD) analysis revealed that the three aptamers formed stable B-form, stem-loop conformations. The selected aptamers were used to construct a chemiluminescence (CL) aptasensor biosensing platform to detect NSE from actual serum samples. Experimental results confirmed that the CL immunosensing platform had good sensitivity with detection limits of 1-100 ng mL-1. The results demonstrated that our obtained the Apt-5 aptsensor was highly specific in the detection of NSE in serum samples. The detection limit was 0.1 ng mL-1, which was lower than the 0.25 ng mL-1 limit of the ELISA used at the hospital. Moreover, the aptasensor can contribute to better detection of small cell lung cancer (SCLC).

DNA aptamers from whole-serum SELEX as new diagnostic agents against gastric cancer.

Gastric cancer is still among the leading causes of cancer deaths worldwide. Despite the improvements in diagnostic methods, the status of early detection has not been achieved so far. Early diagnosis of gastric cancer may significantly improve the cure rate of patients. Therefore, a new diagnostic method is needed. In this study, subtractive SELEX was performed to screen gastric cancer serum-specific DNA aptamers by using gastric cancer serum and normal serum as the target and negative serum, respectively. Four highly specific aptamers generated for gastric cancer serum, Seq-3, Seq-6, Seq-19 and Seq-54, were developed using whole-serum subtractive SELEX technology with K d of 128 ± 26.3 nM, 149 ± 23.6 nM, 232 ± 44.2 nM, 202 ± 25.6 nM, respectively. These generated aptamers showed higher specificities toward their target serum by differentiating normal serum but closely related other cancer serums. The selected four high affinity DNA aptamers were further applied to the development based on qPCR method for the early detection of gastric cancer. In addition, we performed MALDI-TOF MS followed by secondary peptide sequencing MS analysis for the identification of the aptamer binding proteins. Among these potential biomarkers, APOA1, APOA4, PARD3, Importin subunit alpha-1 showed a relatively high score probability. Therefore, these four ssDNA aptamers generated in our study could be a promising molecular probe for gastric cancer diagnosis.