Jingmou Yu

Fabrication and characterization of nuclear localization signal-conjugated glycol chitosan micelles for improving the nuclear delivery of doxorubicin.

Background Supramolecular micelles as drug-delivery vehicles are generally unable to enter the nucleus of nondividing cells. In the work reported here, nuclear localization signal (NLS)-modified polymeric micelles were studied with the aim of improving nuclear drug delivery. Methods In this research, cholesterol-modified glycol chitosan (CHGC) was synthesized. NLS-conjugated CHGC (NCHGC) was synthesized and characterized using proton nuclear magnetic resonance spectroscopy, dynamic light scattering, and fluorescence spectroscopy. Doxorubicin (DOX), an anticancer drug with an intracellular site of action in the nucleus, was chosen as a model drug. DOX-loaded micelles were prepared by an emulsion/solvent evaporation method. The cellular uptake of different DOX formulations was analyzed by flow cytometry and confocal laser scanning microscopy. The cytotoxicity of blank micelles, free DOX, and DOX-loaded micelles in vitro was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in HeLa and HepG2 cells. Results The degree of substitution was 5.9 cholesterol and 3.8 NLS groups per 100 sugar residues of the NCHGC conjugate. The critical aggregation concentration of the NCHGC micelles in aqueous solution was 0.0209 mg/mL. The DOX-loaded NCHGC (DNCHGC) micelles were observed as being almost spherical in shape under transmission electron microscopy, and the size was determined as 248 nm by dynamic light scattering. The DOX-loading content of the DNCHGC micelles was 10.1%. The DOX-loaded micelles showed slow drug-release behavior within 72 hours in vitro. The DNCHGC micelles exhibited greater cellular uptake and higher amounts of DOX in the nuclei of HeLa cells than free DOX and DOX-loaded CHGC (DCHGC) micelles. The half maximal inhibitory concentration (IC50) values of free DOX, DCHGC, and DNCHGC micelles against HepG2 cells were 4.063, 0.591, and 0.171 μg/mL, respectively. Moreover, the IC50 values of free DOX (3.210 μg/mL) and the DCHGC micelles (1.413 μg/mL) against HeLa cells were nearly 6.96- and 3.07-fold (P < 0.01), respectively, higher than the IC50 value of the DNCHGC micelles (0.461 μg/mL). Conclusion The results of this study suggest that novel NCHGC micelles could be a potential carrier for nucleus-targeting delivery.

Folic acid conjugated glycol chitosan micelles for targeted delivery of doxorubicin: preparation and preliminary evaluation in vitro

For folate receptor (FR) targeted anticancer therapy, novel folic acid (FA) conjugated cholesterol-modified glycol chitosan (FCHGC) micelles were synthesized and characterized by 1H NMR, dynamic light scattering, transmission electron microscopy, and fluorescence spectroscopy. The degree of substitution was 1.4 FA groups and 7.7 cholesterol groups per 100 sugar residues of glycol chitosan. The critical aggregation concentration of FCHGC micelles in aqueous solution was 0.0169 mg/ml. The doxorubicin (DOX)-loaded FCHGC (DFCHGC) micelles were prepared by an emulsion/solvent evaporation method. The DFCHGC micelles were almost spherical in shape and their size increased from 282 to 320 nm with the DOX-loading content increasing from 4.53 to 11.4%. DOX released from DOX-loaded micelles displayed sustained release behavior. The targeted micelles encapsulated DOX showed significantly greater cytotoxicity against FR-positive HeLa cells than the nontargeted DOX-loaded micelles and free DOX. These results suggested that FCHGC micelles could be a potential carrier for targeted drug delivery.

Development of dual ligand-targeted polymeric micelles as drug carriers for cancer therapy in vitro and in vivo

Chemotherapy is a major therapeutic approach for cancer patients. The action sites of cancer drugs are intracellular compartments including cytoplasm or nucleus. However, targeting drug delivery into the nucleus of specific tumor cells remains a challenging task. Herein, we developed dual-decorated polymeric micelles with folic acid (FA) and a nuclear localization signal (NLS) for specific tumor-targeted drug delivery. Cholesterol-modified glycol chitosan (CHGC) was synthesized. NLS and FA conjugated CHGC (NFCHGC) micelles were constructed. Doxorubicin (DOX) was chosen as a model anticancer drug and coumarin 6 (C6) was used as a hydrophobic fluorescence probe. The drug-loaded polymeric micelles were prepared and characterized. C6-loaded NFCHGC (C6/NFCHGC) showed efficient intracellular trafficking including endosomal/lysosomal escape and nucleus transportation in folate receptor (FR)-positive KB cells investigated by confocal laser scanning microscopy (CLSM). DOX-loaded NFCHGC (DOX/NFCHGC) exhibited stronger cytotoxicity against KB cells than other DOX formulations. Furthermore, blank polymeric micelles displayed low toxicity and good biocompatibility in vivo. DOX/NFCHGC micelles had the strongest anti-tumor efficacy against KB tumor xenograft models in vivo. These findings demonstrated that NFCHGC micelles were deemed as a potential drug nanocarrier for cancer therapy, especially used in FR-positive tumor cells and nucleus-targeting delivery.

Reduction-sensitive micelles self-assembled from amphiphilic chondroitin sulfate A-deoxycholic acid conjugate for triggered release of doxorubicin

Reduction-sensitive chondroitin sulfate A (CSA)-based micelles were developed. CSA was conjugated with deoxycholic acid (DOCA) via a disulfide linkage. The bioreducible conjugate (CSA-ss-DOCA) can form self-assembled micelles in aqueous medium. The critical micelle concentration (CMC) of CSA-ss-DOCA conjugate is 0.047mg/mL, and its mean diameter is 387nm. The anticancer drug doxorubicin (DOX) was chosen as a model drug, and was effectively encapsulated into the micelles with high loading efficiency. Reduction-sensitive micelles and reduction-insensitive control micelles displayed similar DOX release behavior in phosphate buffered saline (PBS, pH7.4). Notably, DOX release from the reduction-sensitive micelles in vitro was accelerated in the presence of 20mM glutathione-containing PBS environment. Moreover, DOX-loaded CSA-ss-DOCA (CSA-ss-DOCA/DOX) micelles exhibited intracellular reduction-responsive characteristics in human gastric cancer HGC-27 cells determined by confocal laser scanning microscopy (CLSM). Furthermore, CSA-ss-DOCA/DOX micelles demonstrated higher antitumor efficacy than reduction-insensitive control micelles in HGC-27 cells. These results suggested that reduction-sensitive CSA-ss-DOCA micelles had the potential as intracellular targeted carriers of anticancer drugs.

Preparation, Characterization and Evaluation of α-Tocopherol Succinate-Modified Dextran Micelles as Potential Drug Carriers

In the present study, α-tocopherol succinate (TOS) conjugated dextran (Dex-TOS) was synthesized and characterized by fourier transform infrared (FT-IR) spectroscopy, 1H nuclear magnetic resonance (1H NMR), dynamic light scattering (DLS) and fluorescence spectroscopy. Dex-TOS could form nanoscaled micelles in aqueous medium. The critical micelle concentration (CMC) is 0.0034 mg/mL. Doxorubicin (Dox) was selected as a model drug. Dox-loaded Dex-TOS (Dex-TOS/Dox) micelles were prepared by a dialysis method. The size of Dex-TOS/Dox micelles increased from 295 to 325 nm with the Dox-loading content increasing from 4.21% to 8.12%. The Dex-TOS/Dox micelles were almost spherical in shape, as determined by transmission electron microscopy (TEM). In vitro release demonstrated that Dox release from the micelles was in a sustained manner for up to 96 h. The cellular uptake of Dex-TOS/Dox micelles in human nasopharyngeal epidermoid carcinoma (KB) cells is an endocytic process determined by confocal laser scanning microscopy (CLSM). Moreover, Dex-TOS/Dox micelles exhibited comparable cytotoxicity in contrast with doxorubicin hydrochloride. These results suggested that Dex-TOS micelles could be a promising carrier for drug delivery.

Reduction-responsive core-crosslinked micelles based on a glycol chitosan–lipoic acid conjugate for triggered release of doxorubicin

Reduction-responsive core-crosslinked micelles were developed based on a glycol chitosan–lipoic acid (GC–LA) conjugate and used for triggered release of doxorubicin (DOX). The substitution degree of GC–LA was 8.3 lipoic acid groups per 100 sugar units of glycol chitosan. GC–LA could form nanoscaled micelles in aqueous solution, wherein the critical micelle concentration (CMC) of 0.081 mg mL−1 was determined. Furthermore GC–LA micelles can be crosslinked by a catalytic amount of dithiothreitol. The mean diameter of DOX-loaded core-crosslinked GC–LA (DOX-GC–LA/cc) micelles increased from 305 to 408 nm as the DOX-loading content increased from 6.03% to 10.74%. DOX-loaded crosslinked micelles demonstrated obvious reduction-triggered destabilization. DOX release from non-crosslinked GC–LA micelles was 87.6% for up to 96 h, whereas 25.3% of DOX release from DOX-GC–LA/cc micelles was observed in phosphate buffered saline (PBS, pH 7.4). Notably, in the presence of a 20 mM GSH-containing environment, accelerated DOX release from DOX-GC–LA/cc micelles was found. The blank micelles had low cytotoxicity in vitro, and DOX-GC–LA/cc micelles demonstrated intracellular redox-responsive characteristics in A549 cancer cells. These results suggested that GC–LA core-crosslinked micelles could be promising carriers for anticancer drug delivery.

Self-aggregated nanoparticles of linoleic acid-modified glycol chitosan conjugate as delivery vehicles for paclitaxel: preparation, characterization and evaluation.

A series of linoleic acid-modified glycol chitosan (LAGC) conjugates were synthesized and characterized by FTIR and 1H NMR. The effect of the amount of linoleic acid (LA) on the physicochemical properties of LAGC conjugates was investigated. The mean diameters of three LAGC nanoparticles determined by dynamic light scattering ranged from 204 to 289 nm. The critical aggregation concentration values of LAGC conjugates in aqueous solution were 0.0148, 0.0348, and 0.0807 mg/ml, respectively. Paclitaxel (PTX) was physically loaded into the LAGC nanoparticles by a dialysis method. The drug loading content and encapsulation efficiency of PTX-loaded LAGC (PTX-LAGC) nanoparticles increased with an increasing ratio of the hydrophobic LA to hydrophilic glycol chitosan in the conjugates. PTX-LAGC nanoparticles were almost spherical in shape observed by transmission electron microscopy. In vitro release revealed that PTX release from the nanoparticles was reduced as the LA substitution degree of LAGC conjugates increased. Compared with the commercial formulation Taxol, PTX-LAGC-1 nanoparticles exhibited comparable cellular uptake and cytotoxicity against HepG2 cells in vitro. Importantly, PTX-LAGC-1 nanoparticles demonstrated the stronger antitumor efficacy against hepatic H22 tumor-bearing mice than Taxol (p < 0.05). Therefore, glycolipid-like LAGC nanoparticles had a potential as delivery vehicles for tumor therapy.

Insulin protects apoptotic cardiomyocytes from hypoxia/reoxygenation injury through the sphingosine kinase/sphingosine 1-phosphate axis.

Objective Experimental and clinical studies have shown that administration of insulin during reperfusion is cardioprotective, but the mechanisms underlying this effect are still unknown. In this study, the ability of insulin to protect apoptotic cardiomyocytes from hypoxia/reoxygenation injury using the sphingosine kinase/sphingosine 1-phosphate axis was investigated. Methods and Results Rat cardiomyocytes were isolated and subjected to hypoxia and reoxygenation. [γ-32P] ATP was used to assess sphingosine kinase activity. Insulin was found to increase sphingosine kinase activity. Immunocytochemistry and Western blot analysis showed changes in the subcellular location of sphingosine kinase 1 from cytosol to the membrane in cardiomyocytes. Insulin caused cardiomyocytes to accumulate of S1P in a dose-dependent manner. FRET efficiency showed that insulin also transactivates the S1P1 receptor. TUNEL staining showed that administration of insulin during reoxygenation could to reduce the rate of reoxygenation-induced apoptosis, which is a requirement for SphK 1 activity. It also reduced the rate of activation of the S1P receptor and inhibited hypoxia/reoxygenation-induced cell death in cardiomyocytes. Conclusion The sphingosine kinase 1/sphingosine 1-phosphate/S1P receptor axis is one pathway through which insulin protects rat cardiomyocytes from apoptosis induced by hypoxia/reoxygenation injury.

Correction: Preparation, Characterization and Evaluation of α-Tocopherol Succinate-Modified Dextran Micelles as Potential Drug Carriers. Materials 2015, 8, 6685–6696

The authors wish to make the following correction to their paper [1]. [...].