Ethosomes as delivery system for transdermal administration of vinpocetine.

The purpose of the present study was to develop a novel transdermal vinpocetine patch containing a stable formulation and with good entrapment efficiency, and percutaneous absorption which via ethosome. Ethosome was found to be a more efficient delivery carrier with high encapsulation capacities (79.5% +/- 1.8%) and nanometric size (180.7 +/- 1.5 nm). In vitro percutaneous permeation experiments demonstrated that the permeation of vinpocetine through abdominal skin of Sprague Dawley was significantly increased when ethosome was used. The vinpocetine transdermal fluxes from ethosome gel (3.56 +/- 0.13 microg/cm2/h) were 6.72 and 3.10 times higher than that of vinpocetine gel solution and vinpocetine aueous solution, respectively. Furthermore, the AUC(0 --> infinity), and eliminiation half-life by the transdermal administration were significantly higher than those by the intragastric administration (P < 0.01). The study demonstrated that ethosome is a promising vesicular carrier for enhancing percutaneous absorption of vinpocetine.

Cationic submicron emulsions overcome multidrug resistance in SGC7901/VCR cells.

The over-expression of P-glycoprotein (P-gp) is associated with the development of multi-drug resistance (MDR) in cancer cells. In this study, we examined whether cationic submicron emulsions (CSEs) can efficiently deliver hydroxycamptothecin (HCPT) into MDR cells (SGC7901/VCR cells) via electrostatic-mediated endocytosis, thus overcoming MDR. We prepared HCPT-CSEs and rhodamine-123-CSEs (RH-123-CSEs), and examined the in vitro cytotoxic activity of HCPT-CSEs and the intracellular accumulation of HCPT and RH-123 in SGC7901/VCR cells. The HCPT-CSEs significantly increased the intracellular accumulation of HCPT (8.2-fold higher than HCPT-injection) and enhanced cytotoxic activity of HCPT (2.7-fold higher than HCPT-injection with verapamil). The fluorescence microscopic and flow cytometric detection on RH-123 supported the intracellular accumulation effect of CSEs. These results indicate CSEs may enhance drug-CSEs internalization followed by releasing their contents into the cytoplasm (near nuclear), thus lowering P-gp-mediated drug efflux. Furthermore, these in vitro results suggest that CSEs are a potentially useful drug delivery system to circumvent P-gp-mediated MDR of tumor cells.

Preparation and in vitro anticancer activity of oxymatrine mixed micellar nanoparticles.

The aim of this study was to prepare oxymatrine (OMT) mixed micellar nanoparticles to delay release of the drug and enhance its cytotoxicity against cancer cells. A co-solvent evaporation method using lipoid E80, lipoid S75, MPEG-PLA and Poloxamer 188 was chosen to prepare the OMT formulation, and its release characteristics, cytotoxic activity in vitro and physical characteristics were evaluated. The results showed that OMT mixed micellar nanoparticles have sustained release and cytotoxic activity in vitro to the SMMC-7721 cell line.

Pharmacokinetics of hydroxycamptothecin nanosuspensions in rats.

The purpose of the present study was to investigate the pharmacokinetics of hydroxycamptothecin nanosuspensions after intravenous administration in rats. Hydroxycamptothecin injection was studied parallelly. The results showed that AUC(0 --> infinity), MRT, t1/2(alpha) and t1/2(beta) of hydroxycamptothecin nanosuspensions was significantly higher, while their total body clearance was lower than those of hydroxycamptothecin injections. The results indicate that hydroxycamptothecin nanosuspensions significantly increase hydroxycamptothecin blood concentrations and retention within the systemic circulation.

Development and validation of an HPLC method for determination of oleanolic acid content and partition of oleanolic acid in submicron emulsions.

The aim of this study was to develop and validate a simple HPLC method for the quantitative determination of the oleanolic acid (OA) content and partition coefficient of OA in a submicron emulsion-based formulation (SE-OA). A Diamonsil C18 (150 mm x 4.6 mm, 5 microm) column was eluted with a mobile phase consisting of methanol/water (95:5, v/v). The analyses were performed at 35 +/- 1 degrees C with a flow rate of 1.0 mL/min and variable wavelength detector (VWD) at 210 nm. The calibration curve was linear over a concentration range of 2-100 microg/mL with a correlation coefficient of 0.999. The LOD and LOQ were 0.1 and 1 microg/mL, respectively. The individual spike recovery of OA ranged from 99.88 to 100.28%. The percent relative standard deviations (% R.S.D.) of intra-day and inter-day analyses were less than 3.1%. The validation results confirmed that the method is specific, linear, accurate, precise, robust and sensitive for its intended use. The present method was successfully applied to the determination of the OA content and partition coefficient of OA in SE-OA during the early stage of formulation development.

[Effect of dihydroartemisinin on multidrug resistance of human oral squamous cell carcinoma cell line KBV200 by regulating ROS-MAPK pathway].

PURROSE: To investigate the effect of dihydroartemisinin (DHA) on multidrug resistance of human oral squamous cell carcinoma cell line KBV200 and to explore its relationship with ROS-MAPK pathway. METHODS: MTT assay was used to detect the effects of different concentrations of DHA on proliferation of KB and KBV200 cells. MTT assay was used to detect the inhibition rates of cisplatin (DDP), vincristine (VCR), adriamycin (ADM), etoposide (VP-16) on proliferations of KB and KBV200 cells and the changes after DHA addition, combined with ROS inducer 3-AT, ERK inhibitor UO126, JNK inhibitor SP600125, and p38MAPK inhibitor SB203580, respectively, the difference of reversal before and after intervention was observed; the fluorescence intensity of ROS was measured by flow cytometry. SPSS 17.0 software package was used to analyze the data. RESULTS: Compared with KB cells, drug resistances of KBV200 cells to VCR, VP-16 and ADM were significantly increased(P<0.05), after 10, 20, and 30 µg/mL DHA treatment; the IC50 of KBV200 cells to VCR, VP-16 and ADM was decreased significantly in a concentration-dependent manner(P<0.05). Compared with KB cells, ROS fluorescence intensity of KBV200 cells decreased(P<0.05); after DHA treatment, ROS fluorescence intensity increased significantly, IC50 of VCR, VP-16, ADM decreased significantly(P<0.05), which was consistent with ROS promoter effect. Compared with KB cells, the levels of p-ERK/ERK, p-JNK/JNK, p-p38MAPK/p38MAPK in KBV200 cells increased significantly (P<0.05); after DHA treatment, the levels of p-ERK/ERK, p-JNK/JNK, p-p38MAPK/p38MAPK decreased significantly, IC50 of VCR, VP-16, and ADM increased significantly(P<0.05); after adding ERK inhibitor UO126, JNK inhibitor SP600125, and p38MAPK inhibitor SB203580, IC50 of VCR, VP-16, and ADM to KBV200 cells were further reduced. CONCLUSIONS: Dihydroartemisinin may reverse multidrug resistance of KBV2001 cells by promoting ROS production and inhibiting MAPK pathway.

High-performance liquid chromatographic and pharmacokinetic analyses of an intravenous submicron emulsion of perillyl alcohol in rats.

Perillyl alcohol (POH) is currently in phase II clinical trials both as a chemopreventative and chemotherapeutic agent. The present report describes a simple, rapid and sensitive HPLC-UV method to quantify POH in rat plasma. After protein precipitation with acetonitrile, POH was separated using an Agilent Zorbax XDB C(18) column (150 mm x 4.6 mm, 5 microm) with a mobile phase consisting of acetonitrile-water (40:60, v/v) at a flow rate of 1.0 ml min(-1), and detected at 210 nm. The method has been used successfully to determine trace levels of POH in plasma down to 0.015 microg ml(-1). The pharmacokinetics of POH after intravenous administrations in three formulations, i.e. POH solution (POH-SOL), negatively charged submicron emulsions (POH-SE) and positively charged submicron emulsions (POH-CSSE) were investigated. AUC(0-infinity), MRT, t(1/2alpha) and t(1/2beta) of POH-SE and POH-CSSE were significantly higher, while their total body clearance was lower than those of POH-SOL. In addition, AUC(0-infinity), MRT and t(1/2beta) of POH-CSSE were significantly higher than those of POH-SE. The results indicate that the submicron emulsion formulation significantly increases POH blood concentrations and retention within the systemic circulation.

[Prokaryotic Expression and Purification of the Notch Ligand and Its Effect on Hematopoiesis after Carbon Tetrachloride Damage].

OBJECTIVE: To express and purify the mouse endothelial cell-targeted recombinant Notch ligand protein mD1R, and to investigate its effect on hematopoiesis after carbon tetrachloride damage. METHODS: PCR was performed to clone and construct the expression vector pET22b(+)-mD1R. The mD1R successfully transformed into E. coli was induced by IPTG, and purified with Ni2+-beads affinity chromatography. The target protein was detected by SDS-PAGE. The fluorescence-activated cell sorting analysis (FACS), cell adhesion test, immunofluorescence staining and quantitative real-time PCR were employed to detect the endothelial cell-targeted and Notch signaling-activated biological characteristics of mD1R. The carbon tetrachloride mouse model was established to observe the effects of mD1R on the hematopoietic stem cell (HSC), myeloid cells and lymphoid cells by flow cytometry. The Lin-Scal-1+c-Kit+ cells were sorted by magnetic bead, FACS was performed to analyze the cell cycle, and RT-PCR was employed to observe the expression of interleukin (IL)-10. RESULTS: The prokaryotic expression vector was successfully cloned and constructed. The purity and the activity were confirmed in mD1R recombinant protein. The purified mD1R activated the Notch signaling pathway of hematopoietic stem cells in carbon tetrachloride damaged mouse, and internally elevated the number of HSC and long-term HSC to 2.96-fold and 6.18-fold. In addition, mD1R improved the amplification of the myeloid progenitor cells and the myeloid-derived suppressor cells, particularly the granulocyte/monocyte into blood. Mechanistically, the further analyses suggested that Notch pathway could increase the proliferation of HSC and enhance expression of IL-10 after stress injury. CONCLUSIONS: A new and activated recombinant Notch ligand protein has been obtained successfully to communicate hematopoietic stem cells and hematopoietic microenvironment. The Notch- mediated intrinsic hematopoiesis has been regulated by the anti-inflammatory factor after stress injury.

Development and validation of a sensitive reversed-phase HPLC method to determine intracellular accumulation of hydroxycamptothecin.

The intracellular accumulation of anti-cancer agents strongly influences the efficiency of chemotherapy for cancer. In the present study, a simple, rapid, sensitive reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated to determine hydroxycamptothecin (HCPT) in Eca109 cells. HCPT in cellular lysis solution were measured by RP-HPLC with a C18 column after extraction with ethyl acetate. The mobile phase contained 0.1% triethylamine-phosphoric acid buffer (pH 3.0) and acetonitrile (75:25, v/v). Fluorescence detector with excitation and emission wavelengths of 382 and 528 nm was used for determination of HCPT. The calibration curve was linear from 2 to 100 ng/ml with correlation coefficient of 0.9999, while the limit of quantification is 2 ng/ml. The recovery of assay was between 86.5 and 105.2%. The intra- and inter-day coefficients of variation were less than 10% (R.S.D.). Furthermore, the validated method was used to determine the accumulation of HCPT after incubating the liposomal formulation of HCPT and HCPT for injection with the intact cells. HCPT liposomes showed higher intracellular accumulation of HCPT at different incubation times compared with that of conventional HCPT injection.

DNA origami delivery system for cancer therapy with tunable release properties.

In the assembly of DNA nanostructures, the specificity of Watson-Crick base pairing is used to control matter at the nanoscale. Using this technology for drug delivery is a promising route toward the magic bullet concept, as it would allow the realization of complex assemblies that co-localize drugs, targeting ligands and other functionalities in one nanostructure. Anthracyclines' mechanism of action in cancer therapy is to intercalate DNA, and since DNA nanotechnology allows for such a high degree of customization, we hypothesized that this would allow us to tune the DNA nanostructures for optimal delivery of the anthracycline doxorubicin (Dox) to human breast cancer cells. We have tested two DNA origami nanostructures on three different breast cancer cell lines (MDA-MB-231, MDA-MB-468, and MCF-7). The different nanostructures were designed to exhibit varying degrees of global twist, leading to different amounts of relaxation in the DNA double-helix structure. By tuning the nanostructure design we are able to (i) tune the encapsulation efficiency and the release rate of the drug and (ii) increase the cytotoxicity and lower the intracellular elimination rate when compared to free Dox. Enhanced apoptosis induced by the delivery system in breast cancer cells was investigated using flow cytometry. The findings indicate that DNA origami nanostructures represent an efficient delivery system for Dox, resulting in high degrees of internalization and increased induction of programmed cell death in breast cancer cells. In addition, by designing the structures to exhibit different degrees of twist, we are able to rationally control and tailor the drug release kinetics.

In-vivo pharmacokinetics, tissue distribution and anti-tumour effect of hydroxycamptothecin delivered in oil-in-water submicron emulsions.

OBJECTIVES: The aim of this study was to investigate the pharmacokinetics, tissue distribution and anti-tumour effect of hydroxycamptothecin submicron emulsions (HCPT-SEs). METHODS: HCPT-SEs or HCPT injection (HCPT-I) was administered intravenously into the tail vein of rats or S180 tumour-bearing mice. KEY FINDINGS: HCPT-SEs increased the plasma concentration of HCPT compared with HCPT-I at all time points. The AUC(0-∞) , elimination half-life and mean residence time of anionic submicron emulsions containing HCPT (HCPT-ASEs) and cationic submicron emulsions containing HCPT (HCPT-CSEs) were significantly greater than those of HCPT-I (P <0.01). Especially, a prolonged elimination half-life was found for HCPT-CSEs. HCPT-CSEs and HCPT-ASEs resulted in a 7.9-fold and 3.1-fold increase in AUC(0-6h) of tumour compared with HCPT-I, respectively. The targeting efficiency (T(e) ) of HCPT-ASEs and HCPT-CSEs indicated their selectivity to tumour and the T(e) of HCPT-CSEs was significantly higher than that of HCPT-ASEs (P<0.01). The anti-tumour effect studies showed that HCPT-SEs improved the therapeutic efficiency of HCPT compared with HCPT-I. The percentage of tumour growth suppression rate of mice treated with HCPT-CSEs (2.0 mg HCPT eq./kg) increased 2.1 fold compared with that of HCPT-I. CONCLUSIONS: Submicron emulsions can alter the pharmacokinetic characteristics and tissue distribution of HCPT, and enhance tumour targeting and anti-tumour activity.

[Expression of serum sICAM-1 in patients with primary hepatocellular carcinoma and its relationship with liver fibrosis].

AIM: To explore the clinical value of serum soluble intercellular adhesion molecule-1 (sICAM-1) in patients with primary hepatocellular carcinoma (PHC) and its relationship with liver fibrosis. METHODS: The serun sICAM-1, PC III, IV-C, LN and HA level of 45 cases of patients with PHC, 30 cases of benign tumor and 35 cases of healthy people were determined by ELISA, the relationship between sICAM-1 and liver fibrosis was analyzed. RESULTS: The serum sICAM-1, PC III, IV-C, LN and HA levels of the PHC group were significantly higher than that of the benign tumor group and normal control group, compared the difference was significant (P<0.05); The serum markers was no significant difference between the benign tumor group and normal control group (P>0.05); The serum sICAM-1 was positively correlated with the PC III, IV-C, LN and HA (γ= 0.683, 0.575, 0.573 and 0.539, P<0.05). CONCLUSION: Detection of serum sICAM-1 has important clinical significance for assessing the PHC patient's condition, early diagnosing and treating liver cancer.

Preparation and cytotoxic activity of hydroxycamptothecin nanosuspensions.

Hydroxycamptothecin is a promising anticancer agent that possesses the ability to inhibit the growth of a wide range of human tumors. Owing to its poor solubility and instability, the pharmaceutical development and clinical utilization of hydroxycamptothecin have been limited. In the present study, a novel precipitation-combined high-pressure homogenization (PCH) technique was used to prepare hydroxycamptothecin nanosuspensions. Based on the homogenization pressure and number of cycles, the process with 10 cycles at 18,000 psi of homogenization pressure was found to be the most efficient method to achieve consistent particle size reduction. It was used to prepare nanosuspensions for characterization and evaluation of the formulation performance. Lyophilization of hydroxycamptothecin nanosuspensions, the shape and crystal form of the drug, and antiproliferative activity were also studied. The mean particle size (z-ave) of the reconstituted freeze-dried powder was small and uniform. The freeze-dried powder might be a good choice for intravenously administrating poorly soluble hydroxycamptothecin, which proved to have higher cytotoxicity against the cancer cells than hydroxycamptothecin injections (p<0.001). Overall, these studies have demonstrated that the PCH technique can be used successfully to prepare hydroxycamptothecin nanosuspensions.