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Numerous cellular functions occur in spatially and temporally confined regions. Recent studies have shown that membrane-less organelles and compartments in the cell are assembled via liquid–liquid phase separation (LLPS). In vitro LLPS assays using recombinant expressed and purified proteins are necessary for us to further understand how the assembly of phase-separated compartments is regulated in cells. However, uniform standards and protocols are lacking for these In vitro studies. Here, we describe a step-by-step protocol commonly used to investigate In vitro LLPS using purified proteins. This protocol includes expression and purification of the studied proteins, induction of LLPS of the purified proteins, and studies of the biophysical properties of the liquid droplets formed by LLPS. These protocols can be easily followed by researchers to investigate the LLPS behaviors of proteins of interest.
Over the past decades, cell surface charge, although experimentally observed, has not been well understood particularly from the viewpoint of biophysics. Our recent studies have shown that all cancer cells exhibit negative surface charges that are directly proportional to the secreted lactic acid, a unique cancer metabolic characteristic: high rate of glycolysis. We have therefore designed and developed a set of electrically-charged, fluorescent, and super-paramagnetic nanoprobes, capable of sensitive detection of cancer cells based on the surface charges. These probes are utilized to bind onto cells via electrostatic reaction for capture and magnetic separation. In this fashion, we are able to characterize cell surface charges that are regulated by different metabolic patterns, therefore effectively distinguishing the cancer cells from the normal cells. All 22 cancer cells of different organs are found to be negativelycharged therefore bound strongly by the positively-charged nanoprobes, whereas the normal cells show insignificant binding to the nanoprobes of either charge signs (positive or negative). This finding suggests that all tested cancer cells are negatively-charged and normal cells are either charge-neutral or slightly positive. For diagnosis, cancer cells can be detected, electrostatically bound, and magnetically separated in blood by charged and super-paramagnetic nanoprobes. In therapeutics, circulating cancer cells (CTCs) can be filtered and removed in a continuous fashion to reduce the risk of cancer metastasis. If successful, this new nanotechnology will revolutionize early cancer diagnosis and potentially enable new therapeutics in clinical settings.
The adaptive treatment tolerance (ATT) of cancer cells is the main encumbrance to cancer chemotherapy. A potential solution to this problem is to treat cancer cells with multiple drugs using nanoparticles (NPs). In this study, we tested the co-administration of curcumin (Cur) and doxorubicin (Dox) to MCF-7 resistant breast cancer cells to block the ATTand elicit efficient cell killing. Drugs were co-administered to cells both sequentially and simultaneously. Sequential drug co-administration was carried out by pre-treating the cells with albumin nanoparticles (ANPs) loaded with Cur (Cur@ANPs) followed by treatment with Dox-loaded ANPs (Dox@ANPs). Simultaneous drug co-administration was carried out by treating the cells with ANPs loaded with both the drugs (Cur/Dox@ANPs). We found that the simultaneous drug co-administration led to a greater intra-cellular accumulation of Dox and cell killing with respect to the sequential drug co-administration. However, the simultaneous drug co-administration led to a lower intracellular accumulation of Cur with respect to the sequential drug co-administration. We showed that this result was due to the aggregation and entrapment of Cur in the lysosomes as soon as it was released from Cur@ANPs, a phenomenon called lysosomotropism. In contrast, the simultaneous release of Dox and Cur from Cur/Dox@ANPs into the lysosomes led to lysosomal pH elevation, which, in turn, avoided Cur aggregation, led to lysosome swelling and drug release in the cytosol, and finally provoked efficient cell killing. Our study shed the light on the molecular processes driving the therapeutic effects of anti-cancer drugs co-administered to cancer cells in different manners.
The local Ca2+ release from the heterogeneously distributed endoplasmic reticulum (ER) calcium store has a critical role in calcium homeostasis and cellular function. However, single fluorescent proteinbased ER calcium probes experience challenges in quantifying the ER calcium store in differing live cells, and intensity-based measurements make it difficult to detect local calcium microdomains in the ER. Here, we developed a genetically encoded ratiometric ER calcium indicator (GCEPIA1-SNAPER) that can detect the real-time ER calcium store and local calcium microdomains in live cells. GCEPIA1-SNAPER was located in the lumen of the ER and showed a linear, reversible and rapid response to changes in the ER calcium store. The GCEPIA1-SNAPER probe effectively monitored the depletion of the ER calcium store by TG or starvation treatment, and through its use we identified heterogeneously distributed calcium microdomains in the ER which were correlated with the distribution of STIM1 clusters upon ER calcium store depletion. Lastly, GCEPIA1-SNAPER can be used to detect the ER calcium store by highthroughput flow cytometry and confers the ability to study the function of calcium microdomains of the ER.
Systemic toxicity and insufficient drug accumulation at the tumour site are main barriers in chemotherapy. Thermosensitive liposomes (TSL) combined with high intensity focused ultrasound (HIFU) has emerged as a potential solution to overcome these barriers through targeted drug delivery and localised release. Owing to the multiple physical and biochemical processes involved in this combination therapy, mathematical modelling becomes an indispensable tool for detailed analysis of the transport processes and prediction of tumour drug uptake. To this end, a multiphysics model has been developed to simulate the transport of chemotherapy drugs delivered through a combined HIFU–TSL system. All key delivery processes are considered in the model; these include interstitial fluid flow, HIFU acoustics, bioheat transfer, drug release and transport, as well as tumour drug uptake. The capability of the model is demonstrated through its application to a 2-D prostate tumour model reconstructed from magnetic resonance images. Our results not only demonstrate the feasibility of the model to simulate this combination therapy, but also confirm the advantage of HIFU–TSL drug delivery system with enhancement of drug accumulation in tumour regions and reduction of drug availability in normal tissue. This multiphysics modelling framework can serve as a useful tool to assist in the design of HIFU–TSL drug delivery systems and treatment regimen for improved anticancer efficacy.