FMS-like tyrosine kinase 3 (FLT3) is an ideal drug target for the treatment of acute myeloid leukemia (AML). Although several FLT3 inhibitors have been approved or evaluated in clinical trials, selectivity over c-Kit kinase and FLT3 WT remains a major challenge. Herein, we report a series of 4-(2-fluorophenoxy)pyridine derivatives with potent inhibitory activities against FLT3 internal tandem duplication (FLT3-ITD). The representative compound 13v inhibited FLT3-ITD kinase and isogenic BaF3-FLT3-ITD cells with nanomolar IC50 values and achieved selectivity over c-Kit (>53-fold) and FLT WT (19-fold) in transformed BaF3 cells. In addition, compound 13v displayed excellent selectivity against FLT3-ITD driven AML cells compared to other leukemia cells, solid tumors, and normal peripheral blood mononuclear cells. Mechanistic studies revealed that 13v disrupted FLT3 signal transduction and induced G0/G1 cell cycle arrest and apoptosis. Moreover, it also showed good developmental profiles in ADME assays. In in vivo studies, 13v demonstrated desirable pharmacokinetic (PK) profiles and sufficient tumor growth inhibition in a MOLM-13 xenograft model. Taken together, 13v may represent a starting point for the development of improved FLT3-ITD inhibitors.

Core-satellite-structured magnetic nanosorbents (MNs) used for the selective extraction of macrolide antibiotics (MACs) were prepared in this study. The MNs (core-satellite polydopamine-coated Fe3O4 nanoparticles-hollow porous molecularly imprinted polymer) consisted of polydopamine-coated Fe3O4 nanoparticles (Fe3O4@PDA) "core" linked to numerous hollow porous molecularly imprinted polymer (HPMIP) "satellites" with bridging amine functional groups. It is worth mentioning that HPMIPs act as "anchors" for selectively capturing target molecules. Polymers were characterized using TEM, SEM, FT-IR, VSM, and TGA and applied as magnetic dispersive solid-phase extraction (MDSPE) sorbents for the enrichment of trace MACs from a complex food matrix prior to quantification by HPLC-MS/MS. Nanocomposites revealed outstanding magnetic properties (36.1 emu g-1), a high adsorption capacity (103.6 μmol g-1), selectivity (IF = 3.2), and fast kinetic binding (20 min) for MACs. The multiple advantages of the novel core-satellite-structured magnetic molecularly imprinted nanosorbents were confirmed, which makes us believe that the preparation method of the core-satellite MNs can be applied to other fields involving molecular imprinting technology.

Active targeting modification is one of the foremost nanomedicine strategies for the efficacy improvement. Compared to the homogeneous ligandation on spherical nanocarriers, non-spherical nanomedicines usually make the ligand modification more complicated. The modified ligands always exhibit anisotropy and heterogeneity. However, there is very little systematic study on these diversified anisotropic modifications. The efficacy difference and underlying mechanism were still unclear. Here, we separately fabricated hybrid nanodiscs (NDs) conjugated with cRGD on the edge and plane surfaces to engineer two anisotropic targeting nanocarriers (E-cRGD-NDs and P-cRGD-NDs, respectively) for gene delivery. The ligand anisotropy endowed NDs with diversified cellular interactions, and caused different efficacies between E-cRGD-NDs and P-cRGD-NDs. Of note, E-cRGD-NDs showed significant superiority in siRNA loading, cellular uptake, silence efficiency, protein expression and even in vivo efficacy. The mechanism investigation revealed the functional anisotropy specifically for EcRGD-NDs. The edge modification of cRGD efficiently separated the targeting and siRNA loading domains, maximizing their respective functions. These findings reflected the unique effect of ligand anisotropy, also provided a new strategy for the targeting screening of extensive nanomedicines.

Chemotherapy post cancer surgery has important clinical significance for reducing the chance of recurrent-metastasis. However, postoperative chemotherapy efficacy is hampered by poor targeting capability and dose-limiting toxicity of chemo-drugs. Herein, we report a bio-mimetic platelet membrane-cloaked paclitaxel nano-crystal system (PPNCs), which consists of spherical paclitaxel nanocrystals (PNCs) as a high-dose drug core, polyethylene glycol-conjugated paclitaxel (PEG-PTX) as an amphiphilic molecule to adjust the surface hydrophilicity of PNCs and the shell of platelet membrane that can target surgical coagulation site. The in vitro characterization of PPNCs exhibited uniform particle size distribution, high drug loading, and good stability, which are crucial for effective drug delivery. At cell levels, PPNCs showed greater cellular uptake and higher cytotoxicity in 4T1 breast cancer cells than bare PNCs. In vivo, the nanoparticles could deliver high-dose chemodrugs and target the coagulation site caused by surgery or vascular disrupting agents, resulting in enhanced antitumor efficacy and reduced systemic toxicities. In general, the PPNCs system can be served as a promising and efficient drug delivery system for postoperative chemotherapy.

Aurum nanomaterials (ANM), combining the features of nanotechnology and metal elements, have demonstrated enormous potential and aroused great attention on biomedical applications over the past few decades. Particularly, their advantages, such as controllable particle size, flexible surface modification, higher drug loading, good stability and biocompatibility, especially unique optical properties, promote the development of ANM in biomedical field. In this review, we will discuss the advanced preparation process of ANM and summarize their recent applications as well as their prospects in diagnosis and therapy. Besides, multi-functional ANM-based theranostic nanosystems will be introduced in details, including radiotherapy (RT), photothermal therapy (PTT), photodynamic therapy (PDT), immunotherapy (IT), and so on.

Overcoming the epithelial barriers to enhance drug transport is a focused topic for gastrointestinal, intratracheal, intranasal, vaginal, and intrauterine delivery. Nanomedicines with targeting functionization promote such a process owing to specific ligand-receptor interaction. However, compared to the cell uptake of targeting nanotherapies, currently few studies concentrate on their transcytosis including endocytosis for "in" and exocytosis for "out". In fact, the cellular regulatory mechanism for these pathways as well as the principle of ligand's effect on the transcytosis are almost ignored. Here, we fabricated transferrin (Tf) functionalized nanogranules (Tf-NG) as the nanomedicine model and confirmed the difference in polar distributions of Tf receptors (TfRs) between two epithelium models (bipolarity for Caco-2 and unipolarity for MDCK cells). Compared to the nonspecific reference, Tf-conjugation boosted the endocytosis by different pathways in two cell models and transformed the intracellular route of Tf-NG in both cells differently, affecting exocytosis, recycling, and degradation but not the secretion pathway. Only bipolar cells could establish a complete transport flow from "in" to "out", leading to the enhanced transcytosis of Tf-NG. Importantly, epithelia could make responses to Tf-NG transcytosis. Based on the quantitative proteomics, the intracellular trafficking of Tf-NG altered the protein expression profiles, in which the endocytosis- and transcytosis-related proteins were specifically upregulated. Particularly, only bipolar cells could positively feed back to such trafficking via accelerating the subsequent Tf-NG transcytosis. Here, all the cell transport of Tf-NG was polarity associated. In summary, Tf modification elevated the transcytosis of Tf-NG across the epithelium by triggering the polarity-associated transport flow and positive cell feedback loop. These findings provided an insight into the targeting nanodelivery for efficient transport through epithelial barriers.

Gemcitabine (Gem) as an anti-cancer agent has been limited by its short circulation time and rapid metabolism that reflects in low tumor uptake and low therapeutic efficiency. To improve its anti-tumor activity, a novel FAPα enzyme-activated prodrug of Z-GP-Gem modified at 4-amino group of Gem was developed, which could effectively release parent Gem based on the specific cleavage via FAPα enzyme-activation in tumor microenvironment. Compared to Gem, the Z-GP-Gem prodrug exhibited significantly enhanced inhibition of both tumor growth and pulmonary metastasis in BALB/c mice bearing orthotopic breast 4T1 tumors. The Z-GP-Gem prodrug has a prolonged circulation time and a high tumor uptake based on the modification of Z-GP dipeptide at 4-amino group of Gem. These eventually caused a marked improvement in the systemic toxicity and the tumor growth inhibition in 4T1 cells. More interestingly, the unexpected depletion of tumor-associated fibroblast (TAF) was observed during the treatment of Z-GP-Gem prodrug in animal model. Therefore, these findings demonstrated that the FAPα-activated prodrug Z-GP-Gem would be a desirable approach for tumor therapy by intravenous administration.

Introduction Endocytosis of nanomaterials is the first step of nano-bio interaction and current regulation is mostly by nanomaterials but seldom by intracellular signaling proteins. Materials and methods Herein, we synthesized tubular nanocarbon (oxMWCNT) and lamellar-like nanocarbon (oxGRAPHENE) and formulated their aqueous dispersion. A549 and Caco-2 cells were selected as the models of tumor and intestinal epithelial cells, respectively. After knocking down three members of Rho GTPases (Cdc42, Rac1, RhoA) in these two cell lines, their silencing effects on the uptake pathways of nanomaterials with different morphologies were investigated. Results An unexpected finding was that the knock-down led to opposite uptake trends in different types of cells. The endocytosis of carbon nanomaterials increased in Caco-2 cells when Rho GTPases were inactivated, while that in A549 cells decreased. For nanomaterials with different shapes, the involved GTPase member of Rho family, or regulating protein molecule, was different. Concretely, Cdc42 and Rac1 were involved in oxMWCNT endocytosis, while all three GTPases participated in oxGRAPHENE internalization. More interestingly, such difference induced different uptake pathways, namely, the cellular uptake of oxMWCNT was clathrin-mediated and oxGRAPHENE was caveolin-modulated, both with the involvement of dynamin. Conclusion In conclusion, this study provides new insights for the potential intervention in nano-bio interplay.

Gold nanoparticles are promising drug delivery vehicles for nucleic acids, small molecules, and proteins, allowing various modifications on the particle surface. However, the instability and low bioavailability of gold nanoparticles compromise their clinical application. Here, we functionalized gold nanoparticles with CPP fragments (CALNNPFVYLI, CALRRRRRRRR) through sulfhydryl PEG to increase their stability and bioavailability. The resulting gold nanoparticles were characterized with transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-visible spectrometry and X-ray photoelectron spectroscopy (XPS), and the stability in biological solutions was evaluated. Comparing to PEGylated gold nanoparticles, CPP (CALNNPFVYLI, CALRRRRRRRR)-modified gold nanoparticles showed 46 folds increase in cellular uptake in A549 and B16 cell lines, as evidenced by the inductively coupled plasma atomic emission spectroscopy (ICP-AES). The interactions between gold nanoparticles and liposomes indicated CPP-modified gold nanoparticles bind to cell membrane more effectively than PEGylated gold nanoparticles. Surface plasmon resonance (SPR) was used to measure interactions between nanoparticles and the membrane. TEM and uptake inhibitor experiments indicated that the cellular entry of gold nanoparticles was mediated by clathrin and macropinocytosis. Other energy independent endocytosis pathways were also identified. Our work revealed a new strategy to modify gold nanoparticles with CPP and illustrated the cellular uptake pathway of CPP-modified gold nanoparticles.

In this study, monomethoxy (polyethylene glycol)-b-P (d,l-lactic-co-glycolic acid)-b-P (l-glutamic acid) (mPEG-PLGA-PGlu) nanoparticles with the ability to rapidly respond to the endolysosomal pH and hydrolase were prepared and the pH-sensitivity was tuned by adjusting the length of the PGlu segment. The mPEG5k-PLGA20k-PGlu (60) nanoparticles were specifically responsive to an endosomal pH of 5.0-6.0 due to the configuration transition of the PGlu segment and rapidly initiated chemical degradation after incubation with proteinase k for 10 min. Doxorubicin hydrochloride (DOX), used as a model drug, was easily encapsulated into nanoparticles and the DOX-loaded nanoparticles (DOX-NPs) exhibited a pH-dependent and enzyme-sensitive release profile in vitro. The dual sensitivity enabled the rapid escape of DOX-loaded nanoparticles from the endolysosomal system to target cellular nuclei, which resulted in increased cell toxicity against MCF/ADR resistant breast cancer cells and a higher cellular uptake than free DOX. In Vivo Imaging studies indicated that the nanoparticles could continuously accumulate in the tumor tissues through EPR effects and Ex vivo Imaging biodistribution studies indicated that DOX-NPs increased drug penetration into tumors compared with normal tissues. The in vivo antitumor activity demonstrated that DOX-loaded NPs had less body loss and a significant regression of tumor growth, indicating the increased anti-tumor efficacy and lower systemic toxicity. Therefore, this dual sensitive nanoparticle system may be a potential nanocarrier to overcome the multidrug resistance exhibited by breast cancer.

Disulfiram (DSF) has been used to treat alcoholism for many years and it has been suggested to play a key role in combatting many kinds of tumors. However, disulfiram has complex pharmacokinetics and is rapidly eliminated which limits its use as a tumor treatment. Therefore, a rapid and sensitive analytical method based on ultra performance liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) was developed and validated for the determination of disulfiram in rat plasma. Blood samples were pre-stabilized with a stabilizing agent and then plasma was obtained and subjected to solid phase extraction (SPE), and chromatographed on a Phenomenex Kinetex (R) XB C-18 column with gradient elution using a mobile phase consisting of acetonitrile-water (containing 0.1% formic acid and 1 mM ammonium acetate) at a flow rate of 0.2 mL/min for 3 min. Multiple reactions monitoring in positive mode was carried out with disulfiram at 296.95/115.94 and diphenhydramine (internal standard, IS) at 256.14/167.02 over a linear range from 0.6 to 1200 ng/mL. The extraction recovery of disulfiram for different concentrations ranged from 75.7% to 78.3%. The intra- and inter-day precision was less than 8.93% and 12.39%, respectively, and the accuracy was within +/- 7.75%. The validated method was successfully applied to a pharmacokinetic study of disulfiram in rat plasma after oral administration of a dose of 180 mg/kg. (c) 2013 Elsevier B.V. All rights reserved.

Objective To establish an ion-pair RP-HPLC method for the determination of the contents of α-ketoleucine calcium and its related substances.Methods The determination of α-ketoleucine calcium and the separation of its related substances were performed on a C18 column(250 mm×4.6 mm,5 μm).The mobile phase was acetonitrile-0.05%(w)potassium dihydrogen phosphae(pH2.5)(V∶V=23∶67)with 0.01%(w)tetrabutyl ammonium hydroxide.The flow rate was 1.0 mL·min-1.Ultraviolet absorption detector was set at 220 nm with column temperature at 30 ℃.Results The linear range of α-ketoleucine calcium was between 40 and 160 mg·L-1(r=0.999 7).The average recovery was 100.4%(w)with RSD of 0.7%.The related substances of α-ketoleucine calcium could be completely separated from α-ketoleucine calcium.The limit of detection(LOD)was 16 ng.Conclusion The method is simple and accurate for quality control of the α-ketoleucine calcium.

Carbamazepine (CBZ)–hydroxypropyl-β-cyclodextrin (HP-β-CD) complex in the presence of HPMC was prepared and characterized by differential scanning calorimetry (DSC) and X-ray diffractometer intended for improving the dissolution rate of CBZ. The phase-solubility method was used to investigate the effect of HP-β-CD and HPMC on the solubility of CBZ. Tablets of the resulting complex were prepared using direct compression method and the bioavailability was evaluated in beagle dogs using a UPLC/MS/MS method. The results showed solubility of CBZ was increased up to 95 times by complexation with HP-β-CD in the presence of 0.1% HPMC. The results of DSC and X-ray diffraction proved a formation of complex between CBZ and HP-β-CD. Dissolution rate of CBZ was notably improved from complex tablets with more than 97.39% released within 10 min; whereas for the commercial tablets, around 60% was released within 30 min. Using commercial tablets as the reference formulation, the bioavailability of complex tablets was considerably increased by 1.5-fold (P < 0.05) and Tmax was reduced to 0.88 h compared with 1.25 h for commercial tablets. Furthermore, a lower inter-subject variability (49.9%) was observed compared with that of the commercial tablets (39.7%). It is evident from the results herein that complexation with HP-β-CD in the presence of HPMC is a feasible way to prepare a rapidly acting and better absorbed CBZ oral product.