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Smart nanotheranostic systems (SNSs) have attracted extensive attention in antitumor therapy. Nevertheless, constructing SNSs with disease diagnosis ability, improved drug delivery efficiency, inherent imaging performance, and high treatment efficiency remains a scientific challenge. Herein, ultrasmall tin dioxide (SnO

Single cell MS (SCMS) techniques are under rapid development for molecular analysis of individual cells among heterogeneous populations. Lipids are basic cellular constituents playing essential functions in energy storage and the cellular signaling processes of cells. Unsaturated lipids are characterized with one or multiple carbon–carbon double (C=C) bonds, and they are critical for cell functions and human diseases. Characterizing unsaturated lipids in single cells allows for better understanding of metabolomic biomarkers and therapeutic targets of rare cells (e.g., cancer stem cells); however, these studies remain challenging. We developed a new technique using a micropipette needle, in which Paternò-Büchi (PB) reactions at C=C bond can be induced, to determine locations of C=C bonds in unsaturated lipids at the single-cell level. The micropipette needle is produced by combining a pulled glass capillary needle with a fused silica capillary. Cell lysis solvent and PB reagent (acetone or benzophenone) are delivered into the micropipette needle (tip size ≈ 15 um) through a fused silica capillary. The capillary needle plays multiple functions (i.e., single cell sampling probe, cell lysis container, microreactor, and nano-ESI emitter) in the experiments. Both regular (no reaction) and reactive (with PB reaction) SCMS analyses of the same cell can be achieved. C=C bond locations were determined from MS scan and MS/MS of PB products assisted by Python programs. This technique can potentially be used for other reactive SCMS studies to enhance molecular analysis for broad ranges of single cells.

The exploration of single cells reveals cell heterogeneity and biological principle of cellular metabolism. Although a number of mass spectrometry (MS) based single cell MS (SCMS) techniques have been dedicatedly developed with high efficiency and sensitivity, limitations still exist. In this work, we introduced a microscale multifunctional device, the T-probe, which integrates cellular contents extraction and immediate ionization, to implement online in situ SCMS analysis at ambient conditions with minimal sample preparation. With high sensitivity and reproducibility, the T-probe was employed for MS analysis of single HeLa cells under control and anticancer drug treatment conditions. Intracellular species and xenobiotic metabolites were detected, and changes of cellular metabolic profiles induced by drug treatment were measured. Combining SCMS experiments with statistical data analyses, including Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) and two-sample t-test, we provided biological insights into cellular metabolic response to drug treatment. Online MS/MS analysis was conducted at single cell level to identify species of interest, including endogenous metabolites and the drug compound. Using the T-probe SCMS technique combined with comprehensive data analyses, we provide an approach to understanding cellular metabolism and evaluate chemotherapies at the single cell level.

Production of protein therapeutics often involves in vitro refolding from bacterial inclusion bodies and subsequent PEGylation to improve protein stability and plasma half-life. Here, we devised a novel strategy for one-step production of site-specific mono-PEGylated proteins with good bioactivity and improved biostability by integrating PEGylation and protein refolding (IPPR). Using lysozyme and recombinant human fibroblast growth factor 21 (rhFGF21) as model proteins, we showed that both PEGylation and refolding of denatured proteins have been simultaneously accomplished by IPPR with high efficiency of refolding yield and bioconjugation. PEGylated rhFGF21 by IPPR has a similar capacity as the native rhFGF21 to stimulate glucose uptake in 3T3-L1 cells, but exhibits prolonged blood glucose and triglyceride lowering activity levels in the ob/ob diabetic mouse model. Hence, IPPR will significantly facilitate the generation of protein therapeutics.