Anisamide-functionalized pH-responsive amphiphilic chitosan-based paclitaxel micelles for sigma-1 receptor targeted prostate cancer treatment.

Controlled release and tumor-selective distribution are highly desirable for anticancer nanomedicines. Here, we design and synthesize an anisamide-conjugated N-octyl-N,O-maleoyl-O-phosphoryl chitosan (a-OMPC) which can form amphiphilic micelles featuring pH-responsive release and high affinity to sigma-1 receptor-overexpressed tumors for paclitaxel (PTX) delivery. Thereinto, maleoyl and phosphoryl groups cooperatively contribute to pH-responsive drug release due to a conversion from hydrophile to hydrophobe in the acidic microenvironment of endo/lysosomes. We demonstrated that PTX-loaded a-OMPC micelles (PTX-aM) enhanced the cellular internalization via the affinity between anisamide and sigma-1 receptor, rapidly released drug in endo/lysosomes and elevated the cytotoxicity against PC-3 cells. The in vivo studies further verified that PTX-aM could largely accumulate at the tumor site even after 24 h of administration, resulting in obvious inhibition effect and prolonged survival period in PC-3 tumor xenograft-bearing mice. Moreover, OMPC showed no obvious hemolytic and acute toxicity. Collectively, this chitosan derivate holds a promising potential in application of prostate cancer-targeted drug delivery system.

Self-assembled micelles based on N-octyl-N'-phthalyl-O-phosphoryl chitosan derivative as an effective oral carrier of paclitaxel.

Herein, we describe a novel amphipathic chitosan derivative (N-octyl-N'-phthalyl-O-phosphoryl chitosan, abbreviated as OPPC) as an effective oral delivery platform for P-gp substrates, especially paclitaxel (PTX). OPPC could readily self-assemble into micelles, solubilize and encapsulate PTX into the hydrophobic inner core of OPPC with superior loading capacity to chitosan. PTX/OPPC micelles possessed improved intestinal epithelial permeability and oral bioavailability of PTX evaluated by in situ perfusion and pharmacokinetic studies. In vivo fluorescence imaging revealed enhanced stability and integrity of OPPC micelles in mice gastrointestine. Furthermore, cellular uptake studies revealed effective transport and accumulation of OPPC micelles loading PTX or rhodamine-123 into Caco-2 cells via clathrin/cavelin-mediated endocytosis and OPPC-mediated P-gp inhibition. Mechanistically, the inhibition of P-gp efflux pumps by OPPC resulted from the reduction of membrane fluidity and decreased P-gp ATPase activity. In summary, OPPC micelles may serve as an efficient and promising delivery system for enhancing oral bioavailability of P-gp substrates.

Preliminary evidence for the dedifferentiation of RAW 264.7 cells into mesenchymal progenitor-like cells by a purine analog.

Dedifferentiation of cells to multipotential cells is of interest since they have a potential regenerative capacity. Our purpose was to de- and redifferentiate murine RAW 264.7 cells, a committed macrophage cell line of hematopoietic origin, into mesenchymal-like cells such as osteoblasts. RAW 264.7 cells in culture were treated with 5 µM reversine, a purine analog that was shown to dedifferentiate myoblasts in osteoblasts. Treatment with reversine resulted in a significant increase in the expression of the STRO-1 antigen, a marker of mesenchymal stem/progenitor cells: from 0.6%±0.5% cells in untreated RAW cells to 19.0%±8.6% in treated cells, but there was no increase in the expression of SH-2 (CD105), an earlier marker of mesenchymal stem cells. The effects of reversine were significantly curtailed by 67% when cultures were pretreated with the c-Jun N-terminal kinase pathway blocker SP600125. These STRO-1+ cells retained a multipotential status and were capable of redifferentiating into cells with osteogenic and lipogenic characteristics under inductive conditions. We showed that STRO-1+ cells in an osteogenic medium significantly increased expression of the osteoblast marker osteocalcin, and formed mineralized nodules. When seeded on a demineralized scaffold of human bone in vitro, these cells deposited a calcium matrix. Under adipogenic conditions, expression of the adipocyte marker peroxisome proliferator-activated receptor gamma 2 on STRO-1+ cells was elevated, and cultures stained positive with Oil red O. Our results demonstrated that treating a committed hematopoietic cell line with a purine analog can alter cell development and result in cellular reverse transformation into stage-limited multipotential cells. These cells could subsequently be redifferentiated into cells with characteristics of the mesenchymal lineage, such as those of an osteoblast and/or adipocyte, under inductive conditions.

N-octyl-O-sulfate chitosan-modified liposomes for delivery of docetaxel: preparation, characterization, and pharmacokinetics.

A N-octyl-O-sulfate chitosan (NOSC) anchored liposome system was developed as the carrier for antitumor drug, docetaxel (DTX). The physicochemical and pharmacokinetic properties of NOSC-modified DTX liposomes (NDLs) were evaluated compared with the conventional DTX liposomes (DLs) and commercial dosage form of DTX, Taxotere(®). The results showed that NDLs had DTX-loading rate of 3.41%, entrapment efficiency of 61.73%, narrow distributed particle size of 147.6±1.9nm, and high zata potential of -44.2±3.9mV. The decreased permeability of the liposome bilayer was evaluated by release behavior of calcein (CAL) from the internal phase of NOSC-modified CAL liposomes (NCLs) and enhanced stability of NDLs owed to shielding effect of sulfonic shell from adsorption by BSA. After i.v. administration at the dose of 12mg/kg, a significant increase in the AUC, MRT, and T(1/2ß) (P<0.05) was observed in NDLs group compared with DLs and Taxotere(®) group. AUC(0-∞) of NDLs was 6.14 and 1.55 times higher than Taxotere(®) and DLs, respectively, and MRT(0-∞) of NDLs was 5.77 and 1.37 times higher than Taxotere(®) and DLs, respectively. All these results suggested that anchored liposomes could increase the stability of DTX in vitro and in vivo, as compared with conventional liposomes and Taxotere(®). Therefore, NOSC as a polymeric shell to liposomes was effective to enhance the stability of liposomes containing DTX.

Modified chitosan derivative micelle system for natural anti-tumor product gambogic acid delivery.

A chitosan derivative micelle system was developed as the delivery system for a novel anti-tumor drug, gambogic acid (GA). The physicochemical and pharmaceutical properties of GA-loaded micelles (GA-M) were evaluated compared with the formulation GA-L-arginine (GA-L) injection, which entered phase I clinical trials. The results showed that GA-M had high GA-loading rate (29.8 +/- 0.17%), high entrapment efficiency (63.8 +/- 0.52%), and small particle size (108.2 +/- 0.8 nm). After i.v. administration at the dose of 4 mg/kg, the area under concentration-time curve (AUC) and elimination half-life (T(1/2)beta) of GA-M were all increased by 1.7-fold compared with GA-L in rat. Biodistribution study indicated that approximately 67% of GA in the GA-M group was distributed in the liver, while the value of the GA-L group was approximately 55%. Additionally, GA amount in the kidney was greatly reduced in the GA-M group. Also, GA-M was shown to reduce the acute toxicity after i.v. administration in mice compared with GA-L. The present study indicated that GA was rapidly eliminated from the blood and transferred to the tissues, especially the liver. Moreover, GA acute toxicity and irritation to vein were decreased.

PEG conjugated N-octyl-O-sulfate chitosan micelles for delivery of paclitaxel: in vitro characterization and in vivo evaluation.

A series of novel chitosan derivatives (mPEGOSC) with hydrophobic moieties of octyl and hydrophilic moieties of sulfate and polyethylene glycol monomethyl ether (mPEG) groups were synthesized. The values of critical micelle concentration (CMC) were found to be 0.011-0.079 mg/ml, and the log CMC was linearly relative to four structure parameters, degree of substitution (DS) of chitosan unit, sulfate group, PEG unit and octyl group by mole per kilogram. Paclitaxel (PTX)-loaded micelles were prepared with the highest loading rate of 42.6%. In vivo-in vitro correlations of PTX-loaded micelles was designed and conducted, including interaction of the drug-loaded micelles with protein and the kinetics of PTX-loaded micelles below CMC. mPEG2000 and mPEG5000 on the surface of micelles provided stronger inhibition to protein adsorption compared with mPEG1100, and the micelles based on mPEGOSC with higher DS of chitosan unit (mol/kg) shown slower dissociation when diluted below CMC. The pharmacokinetic research of PTX-loaded micelles based on OSC (PTX-OSC), PTX-loaded micelles based on mPEGOSC (PTX-mPEGOSC) and Taxol was carried out in rat. The area under the plasma concentration time curve (AUC (0-t(n))) of PTX-mPEGOSC2000M was 10.21+/-2.12 microg h/ml, which were 2.93 times higher than PTX-OSC and 0.83 times lower than Taxol. Rate constant of distribution phase (alpha) and rate constant of elimination phase (beta) were analyzed by linear regression with the absorbance of protein to micelles (A) and the fraction of intact micelles at 10 min (f(t, 10 min)). The tissue distribution studies in mice indicated that PTX-mPEGOSC2000M micelles were phagocytized less than PTX-OSC micelles by reticuloendothelial system (RES). Furthermore, higher targeting efficiency of PTX-mPEGOSC2000M to uterus (including ovary) was estimated. On the basis of these results, it could be a promising PTX-encapsulated formulation for the chemotherapy of ovarian cancer.

Trabecular microstructure at the human scaphoid nonunion.

PURPOSE: Scaphoid fractures have the highest prevalence of nonunion in the human body, but despite this, little is known about the microstructure of the bone. Given that microstructural changes in the bone at the site of nonunion define the nonunion, it was our purpose to characterize the trabecular microarchitecture on both sides of the nonunion and compare these to the normal structure. METHODS: Fifteen human scaphoid nonunions (14 men, 1 woman; average age, 29 years; interval between injury and examination ranged from 7 to 156 months) were excised during surgical reconstruction and assessed by high-resolution micro-computed tomography. RESULTS: The bone from the nonunions was clearly different from that of normal scaphoids (obtained from nonreplantable hand amputations or postmortem). Bone on both sides of the nonunions was characterized by higher bone mineral density and an increased number of trabeculae, which were thinner and more tightly packed together compared with the normal scaphoid bones. Further evaluation showed that the bone on the proximal side of the nonunion had higher bone mineral density, higher bone volume fraction, decreased bone surface area, increased trabecular thickness, and increased trabecular number when compared with the distal side. CONCLUSIONS: These data demonstrate the differing microstructure of the bone at the scaphoid nonunion, particularly on the proximal side, and indicate that the biology also differs from normal. The data also show that scaphoid nonunions produce new bone at the site, which may have positive implications toward hardware fixation and healing stimulation.

Biological evaluation of N-octyl-O-sulfate chitosan as a new nano-carrier of intravenous drugs.

An amphiphilic chitosan derivate, N-octyl-O-sulfate chitosan (NOSC) was prepared by octylation of amino group at C-2 position and sulfonylation at C-6 position. Micelle formed by NOSC has great capability in solubilization of water-insoluble drug paclitaxel. Enormous attention was attracted by the potential application of NOSC as a new drug delivery system. Tritium labeled NOSC ((3)H NOSC) was injected by tail vein at dose of 13.44 mg/kg in mice; kidney retained the maximum amount of NOSC all the time even after 24h following the injection. Pharmacokinetic parameters (the area under the plasma concentration-time curve, maximum plasma concentration, apparent plasma half-life of distribution phase and elimination phase, mean residence time, apparent volume of distribution, total body clearance) were obtained by fluorometric method in rats. The results showed a linear pharmacokinetics proceeding of FITC-NOSC in vivo. 75.4+/-11.6% (3)H NOSC of dose was excreted in urine over a 7-day period, urinary excretion was the predominant way of excretion of NOSC compared with bilary or fecal pathway. A series of safety studies consisted of acute toxicity study, intravenous stimulation study, injection anaphylaxis study, hemolysis study and cell viability assay were performed to warrant the biocompatibility of the NOSC as intravenous materials. The LD(50) value of NOSC administrated by i.v. and i.p. were calculated as 102.59 and 130.53 mg/kg, respectively. No intravenous stimulation, injection anaphylaxis, hemolysis and cytotoxicity were observed in the safety studies. The tissue distribution, pharmacokinetics, excretion and safety study were persuasive for the potential application of NOSC as a new drug carrier.

Pharmacokinetics, biodistribution, efficacy and safety of N-octyl-O-sulfate chitosan micelles loaded with paclitaxel.

Paclitaxel (Taxol), PTX) is a promising anti-cancer drug and has been successfully used to treat a wide variety of cancers. Unfortunately, serious clinical side effects are associated with it, which are caused by PTX itself and non-aqueous vehicle containing Cremophor EL. Development of new formulation of PTX with better efficacy and fewer side effects is extremely urgent. In the present study, a N-octyl-O-sulfate chitosan (NOSC) micelle was developed and used as the delivery system for PTX. The pharmacokinetics, biodistribution, efficacy and safety of PTX-loaded NOSC micelles (PTX-M) were evaluated. The results showed that NOSC micelles had high drug loading capacity (69.9%) and entrapment efficiency (97.26%). The plasma AUC of PTX-M was 3.6-fold lower than that of Taxol; but the V(d) and CL of PTX-M were increased by 5.7 and 3.5-fold, respectively. Biodistribution study indicated that most of the PTX were distributed in liver, kidney, spleen, and lung and the longest retention effect was observed in the lung. Drug safety assessment studies including acute toxicity, hemolysis test, intravenous stimulation and injection anaphylaxis revealed that the PTX-M was safe for intravenous injection. Furthermore, the comparable antitumor efficacy of PTX-M and Taxol was observed at the same dose of 10 mg/kg in in vivo antitumor mice models inoculated with sarcoma180, enrich solid carcinoma (EC), hepatoma solidity (Heps), Lewis lung cancer cells and A-549 human lung cancer cells. These results clearly showed that PTX-M had the similar antitumor efficacy as Taxol, but significantly reduced the toxicity and improved the bioavailability of PTX.

Role of osterix in endothelin-1-induced downregulation of vascular endothelial growth factor in osteoblastic cells.

Endothelin-1 (ET-1) is produced by vascular endothelial cells to play an important role during bone development, remodeling and repair. ET-1 promotes osteoblastic cell proliferation and differentiation, but has the unique effect of downregulating vascular endothelial growth factor (VEGF) and may thereby control angiogenesis during bone production. Our objectives were to identify the intracellular mechanisms by which ET-1 controls VEGF expression during osteoblastic proliferation and differentiation. ET-1 induced osteoblastic differentiation in rat SBMC-D8 osteoblastic cells, but downregulated expression of VEGF mRNA isoforms (VEGF120, 164 and 188) as demonstrated by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and by use of a luciferase reporter construct containing the promoter region of the VEGF gene. Co-transfection with the endothelin receptor A (ETRA) had the same effect. ET-1 and ETRA both upregulated the transcription factor osterix (Osx). RNA silencing of Osx resulted in an upregulation of VEGF. This study supports the novel inhibitory role for ET-1, via Osx, on VEGF synthesis in osteoblastic cells as a possible mechanism in the temporal and spatial feedback of angiogenesis to bone formation and resorption.

A heterotrimeric death domain complex in Toll signaling.

Signaling from the transmembrane receptor Toll to Rel-related transcription factors regulates dorsoventral patterning of the Drosophila embryo, as well as larval and adult immunity. To identify additional pathway components, we have used double-stranded RNA interference to investigate Drosophila counterparts of genes that regulate the mammalian Rel family member NF-kappaB. Experiments in cultured cells reveal that the fly orthologue of the adaptor protein MyD88 is essential for signal transduction from Toll to a second adaptor protein, Tube. By using coimmunoprecipitation studies, we find a heterotrimeric association of the death domains of MyD88, Tube, and the protein kinase Pelle. Site-directed mutational analyses of interaction sites defined by crystallographic studies demonstrate that Tube recruits MyD88 and Pelle into the heterotrimer by two distinct binding surfaces on the Tube death domain. Furthermore, functional assays confirm that the formation of this heterotrimer is critical for signal transduction by the Toll pathway.