Carbon Dots with Guanidinium and Amino Acid Functional Groups for Targeted Small Interfering RNA Delivery toward Tumor Gene Therapy.

Small interfering RNA (siRNA)-based gene therapy represents a promising strategy for tumor treatment. Novel gene vectors that can achieve targeted delivery of siRNA to the tumor cells without causing any side effects are urgently needed. To this end, the large amino acid mimicking carbon dots with guanidinium functionalization (LAAM GUA-CDs) are designed and synthesized by choosing arginine and dopamine hydrochloride as precursors. LAAM GUA-CDs can load siRNA through the multiple hydrogen bonds between their guanidinium groups and phosphate groups in siRNA. Meanwhile, the amino acid groups at the edges of LAAM GUA-CDs endow them the capacity to target tumors. After loading siBcl-2 as a therapeutic agent, LAAM GUA-CDs/siBcl-2 has a high tumor inhibition rate of up to 68%, which is twice more than that of commercial Lipofectamine 2000. Furthermore, LAAM GUA-CDs do not cause side effect during antitumor treatment owing to their high tumor-targeting ability, thus providing a versatile strategy for tumor-targeted siRNA delivery and cancer therapy.

Multilayer Ratiometric Fluorescent Nanomachines for Imaging mRNA in Live Cells.

Detection of mRNA expression in live cells during treatment is a challenging task, despite its importance in tumor biology and potential therapeutic leads. Here a multilayer ratiometric fluorescent nanomachine for live-cell perturbation and imaging of mRNA at single cell resolution is reported. The nanomachines fabricated by microfluidic approaches consist of fluorescent polymeric cores and multiple lipid layers, which can efficiently deliver siRNA and molecular beacons (MBs) to cytosol and then release the cargo in a sequential way. The siRNA molecules released from the outer lipid layers lead to silencing of multidrug resistance 1 (MDR1) gene, and the MBs from the middle lipid layers detect the presence of MDR1 mRNA. The fluorescent ratio of MBs to fluorescent polymeric cores positively correlates with the expression level of MDR1 mRNA in MCF-7/ADR cells during siRNA treatment. The nanomachines provide comparable results with traditional qPCR for quantifying mRNA, showing great potential for modulation and imaging of intratumoral mRNA in vitro and in vivo.

Improving Tumor Targeting of Exosomal Membrane-Coated Polymeric Nanoparticles by Conjugation with Aptamers.

The coating of natural cellular or exosomal membranes (EMs) onto polymeric nanoparticles has become essential in extending the circulation half-time of nanoparticles by escaping from immune surveillance. Here we report on the surface modification of EM-coated poly(lactic-co-glycolic acid) (PLGA) nanoparticles by AS1411 aptamers (AS-EP) for improved tumor targeting. The combination of microfluidic sonication and cholesterol-modified aptamer functionalization allows for assembly of AS-EP within 10 min. The resulting AS-EP shows a prolonged in vivo circulation time benefiting from the natural properties of exosomes and exhibits high tumor targeting efficiency through specific binding of AS1411 aptamers to nucleolin on the membrane of tumor cells. Moreover, intravenous administration of AS-EP to mice will not result in abnormal pathology or hemolysis. This work opens up opportunities to fabricate and functionalize biomembrane-coated nanoparticles for targeted drug delivery applications.

Microfluidic Sonication To Assemble Exosome Membrane-Coated Nanoparticles for Immune Evasion-Mediated Targeting.

Using natural membranes to coat nanoparticles (NPs) provides an efficient means to reduce the immune clearance of NPs and improve their tumor-specific targeting. However, fabrication of these drug-loaded biomimetic NPs, such as exosome membrane (EM)- or cancer cell membrane (CCM)-coated poly(lactic-co-glycolic acid) (PLGA) NPs, remains a challenging task owing to the heterogeneous nature of biomembranes and labor-intensive procedures. Herein, we report a microfluidic sonication approach to produce EM-, CCM-, and lipid-coated PLGA NPs encapsulated with imaging agents in a one-step and straightforward manner. Tumor cell-derived EM-coated PLGA NPs consisting of both endosomal and plasma membrane proteins show superior homotypic targeting as compared to CCM-PLGA NPs of similar sizes and core-shell structures in both in vitro and in vivo models. The underlying mechanism is associated with a significantly reduced uptake of EM-PLGA NPs by macrophages and peripheral blood monocytes, revealing an immune evasion-mediated targeting of EM-PLGA NPs to homologous tumors. Overall, this work illustrates the promise of using microfluidic sonication approach to fabricate biomimetic NPs for better biocompatibility and targeting efficacy.

Low-cost thermophoretic profiling of extracellular-vesicle surface proteins for the early detection and classification of cancers.

Non-invasive assays for early cancer screening are hampered by challenges in the isolation and profiling of circulating biomarkers. Here, we show that surface proteins from serum extracellular vesicles labelled with a panel of seven fluorescent aptamers can be profiled, via thermophoretic enrichment and linear discriminant analysis, for cancer detection and classification. In a cohort of 102 patients, including 6 cancer types at stages I-IV, the assay detected stage I cancers with 95% sensitivity (95% confidence interval (CI): 74-100%) and 100% specificity (95% CI: 80-100%), and classified the cancer type with an overall accuracy of 68% (95% CI: 59-77%). For patients who underwent prostate biopsies, the assay was superior to the analysis of prostate-specific antigen levels (area under the curve: 0.94 versus 0.68; 33 patients) for the discrimination of prostate cancer and benign prostate enlargement, and also in the assessment of biochemical cancer recurrence after radical prostatectomy. The assay is inexpensive, fast, and requires small serum volumes (<1 µl), and if validated in larger cohorts may facilitate cancer screening, classification and monitoring.

Instrument-free enrichment and detection of phosphopeptides using paper-based Phos-PAD.

The facile detection of phosphopeptides is important for clinical screening and phosphoproteomic research. This work develops an instrument-free, cost-effective, convenient paper-based method for quantitative analysis of phosphorylated peptides. With a novel portable device, Phos-PAD, this method can achieve selective enrichment and colorimetric detection of phosphopeptides within 15 min TiO2 nanoparticle-based chemisorption and tetrabromophenol blue-based colorimetric assay were integrated into the single paper-based analytical device. The color change can indicate the presence of phosphopeptides and the mean pixel intensity of the red channel can be used for phosphopeptide quantification. With capability of quantifying phosphopeptides in serum samples, this Phos-PAD assisted phosphopeptide assay may attract significant attention to clinical analysis of endogenous serum phosphopeptides.

λ-DNA- and Aptamer-Mediated Sorting and Analysis of Extracellular Vesicles.

Extracellular vesicles (EVs) are heavily implicated in diverse pathological processes. Due to their small size, distinct biogenesis, and heterogeneous marker expression, isolation and detection of single EV subpopulations are difficult. Here, we develop a λ-DNA- and aptamer-mediated approach allowing for simultaneous size-selective separation and surface protein analysis of individual EVs. Using a machine learning algorithm to EV signature based on their size and marker expression, we demonstrate that the isolated microvesicles are more efficient than exosomes and apoptotic bodies in discriminating breast cell lines and Stage II breast cancer patients with varied immunohistochemical expression of HER2. Our method provides an important tool to assess the EV heterogeneity at the single EV level with potential value in clinical diagnostics.

Label-free isolation of rare tumor cells from untreated whole blood by interfacial viscoelastic microfluidics.

Label-free, high-throughput, and efficient separation and enrichment of rare tumor cells, such as circulating tumor cells (CTCs), from untreated whole blood is a challenging task, owing to extremely rare events of CTCs and an enormous amount of blood cells. Current strategies for CTC separation always require pre-processing steps including lysis of blood or labeling of CTCs, leading to loss or damage of CTCs. Here, we report an interfacial viscoelastic microfluidic system for size-selective separation of tumor cells directly from whole blood, without the need of cell labeling and other treatments. The sharp flow interfaces between the sample flow and viscoelastic flow (0.05% PEO solutions) in the straight microchannel allow for the penetration of large tumor cells while blocking small blood cells, through exploiting the competition between the inertial lift forces and interfacial elastic lift forces. The microfluidic paradigm does not involve external force fields or complicated fabrication procedures, while achieving 95.1% separation efficiency and 77.5% recovery rate for isolating as few as 50 tumor cells in 1 mL whole blood. The viability of tumor cells after separation is ∼100%, and normal proliferation of separated tumor cells is observed. The interfacial viscoelastic microfluidics holds great promise to facilitate the fundamental and clinical studies of CTCs.