No significant difference in bleeding time was detected in PMDV-coated Si particles or TRAIL-conjugated PMDV-coated Si particles relative to vehicle control (TBS) (Supplemental figure 7e). recognizing a broader spectrum of cancer cells regardless of genetic differences or tumor types. The blood circulation, however, where CTCs transit through, lacks the same tumor microenvironment as that found in a solid tumor. In this study, a unique microenvironment was confirmed upon introduction of cancer cells of different types into circulation where activated platelets and fibrin were physically associated with blood-borne cancer cells. Inspired by this observation, synthetic silica particles were functionalized with activated platelet membrane along with surface conjugation of tumor-specific Tofacitinib apoptosis-inducing ligand cytokine, TRAIL. Biomimetic synthetic particles incorporated into CTC-associated micro-thrombi in lung vasculature and dramatically decreased lung metastases in a Tofacitinib mouse breast cancer metastasis model. Our results demonstrate a Trojan Horse strategy of neutralizing CTCs to Tofacitinib attenuate metastasis. work was produced and purified as previously described . The following chemicals or kits were used for assaying cell proliferation and apoptosis: MTT (AMRESCO, Solon, OH, USA) and TACS? Annexin V-FITC Kit (Gaithersburg, MD, USA). Reagents for SEM and TEM were obtained from Electron Microscopy Sciences (Hatfield, PA, USA): glutaraldehyde, osmium tetroxide and uranyl acetate. APC-conjugated antibodies specific for the extracellular domains of human CD41, CD42b, CD47, CD61 and CD62P in flow cytometry and fluorescence microscopy studies were purchased from Biolegend (San Diego, CA, USA). Primary CD41 antibodies for the extracellular domain (M-148) and cytoplasmic domain (B-9) detection and human CD47 blocking antibody (B6H12) were from Santa Cruz Biotech (Dallas, TX, USA). Synthesis of silica particles Monodisperse silica (Si) particles with a Tofacitinib diameter of 2C3 m were synthesized using tetraethyl orthosilicate (TEOS), 29% ammonia and 100% ethanol via the St?ber method. To produce a positively charged surface, Si particles were suspended in ethanol containing 1 mg/ml 3-aminopropyl triethoxysilane (APTES) and stirred overnight. To prepare FITC-labeled Si particles, FITC was first reacted with APTES in the presence of ethanol and ammonia. Afterwards, TEOS was added to FITC dye solution and stirred overnight to form FITC-labeled Si particles. All synthesized Si particles were washed three times with 100% ethanol followed by three times with TBS to remove free substrate. Particles were characterized with dynamic light scattering using a Zetasizer (Malvern Instruments, Malvern, Worcestershire, UK) and LEO 1550 FE-SEM (Zeiss, Atlanta, GA, USA) prior to PMDV coating. Preparation and functionalization of PMDVs to Si particles Platelets were pelleted from platelet-rich plasma (PRP) through differential centrifugation of whole blood. Following three washes to remove plasma proteins, the isolated platelets were fragmented by seven freeze-thaw cycles and sonication to release platelet membrane-derived vesicles. Then, ultracentrifugation with a discontinuous sucrose gradient (5%, 40%, 55%) was performed to separate membrane vesicles from free proteins, intact platelets, and high-density granules. Previous studies have examined the electrostatically mediated deposition and fusion of negatively charged liposomes on cationic particle supports [27, 28]. In light of the negative surface charge of PMDVs, Si particles with diameters close to platelet size were functionalized with (3-Aminopropyl) triethoxysilane (APTES) to produce a positive charge on the surface. Subsequently, PMDVs were immobilized on the positively charged particle surface by incubating 100 g PMDVs with 10 million particles. After removing free vesicles from the mixture, the coated particles were characterized by dynamic light scattering and electron microscopy. Membrane protein profiling by LC-MS PMDV-coated particles were washed three times with TBS. On-bead tryptic proteolysis protocol was performed. Briefly, proteins were reduced by adding 5 mM DTT (45 min, 56C), and free cysteines were alky lated with iodoacetamide (15 mM, 25C, 1 hr in the dark). A sample of 0.2 g porcine sequencing grade trypsin (Promega, Mannheim, Germany) were added and the samples were incubated overnight at 37C. After digestion, the r eaction was stopped with 10 L of 10% formic acid (FA). The resulting precipitate and particles were removed by centrifugation (13,000 Rabbit Polyclonal to XRCC5 x g, 15 min, 4C). Supernata nt was transferred for LC-MS analysis. Capillary liquid chromatography of tryptic peptides was performed with UltiMate? 3000 RSLCnano LC system (Thermo, Chelmsford, MA, USA). Mass spectrometry analysis of tryptic peptides was performed using Orbitrap Elite (Thermo). Flow cytometry and fluorescence microscopy PMDV-coated and uncoated Si particles were suspended at a concentration of 1 1 million per 100 L blocking buffer PBS/1% BSA. APC-conjugated primary antibodies were added in the blocking buffer and incubated for 30 min at room temperature. Following three washes with 1 mL of PBS, fluorescence measurements were collected using a Guava flow cytometer (EMD, Billerica, MA, USA). Data were analyzed.
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