Facile fabrication of redox-responsive thiol-containing drug delivery system via RAFT polymerization.

A novel kind of redox-responsive polymeric drug delivery system has been designed and prepared successfully through the coupling of the multithiol branched polymers and thiol-containing drugs. The branched poly((S-(4-vinyl) benzyl S'-propyltrithiocarbonate)-co-(poly(ethylene glycol) methacrylate)) (poly(VBPT-co-PEGMA)) was synthesized by one-pot reaction via reversible addition-fragmentation chain transfer (RAFT) copolymerization. Subsequently, the hydrophobic thiol-containing anticancer drug 6-mercaptopurine (MP) was conjugated to poly(VBPT-co-PEGMA) by thiol-disulfide exchange reaction, resulting in the formation of poly(VBPT-co-PEGMA)-S-S-MP conjugate. Due to its amphiphilicity, poly(VBPT-co-PEGMA)-S-S-MP conjugate self-assembled into amphiphilic micelles in aqueous solution. Under a reductive environment, the disassembly of polymeric micelles resulted in the MP release. Flow cytometry and confocal laser scanning microscopy (CLSM) measurements demonstrated that the poly(VBPT-co-PEGMA)-S-S-MP micelles could be taken up by Raji cells (a Burkitt lymphoma cell line). The viability of the Raji cells incubated with the glutathione (GSH) mediated poly(VBPT-co-PEGMA)-S-S-MP micelles was investigated by Cell Counting Kit-8 (CCK-8) assay. The experimental results showed that the viability of the glutathione monoester (GSH-OEt) pretreated cells was lower than that without pretreatment, while the viability of the buthionine sulfoximine (BSO) pretreated cells was higher than that without pretreatment. The poly(VBPT-co-PEGMA)-S-S-MP micelles could induce the apoptosis of Raji cells, and the apoptosis behavior was dose-dependent. This redox-responsive polymer-drug conjugate provides a promising platform for the delivery of thiol-containing biological molecules.

Release behavior and toxicity profiles towards leukemia (WEHI-3B) cell lines of 6-mercaptopurine-PEG-coated magnetite nanoparticles delivery system.

The coating of an active drug, 6-mercaptopurine, into the iron oxide nanoparticles-polyethylene glycol (FNPs-PEG) in order to form a new nanocomposite, FPEGMP-2, was accomplished using coprecipitation technique. The resulting nanosized with a narrow size distribution magnetic polymeric particles show the superparamagnetic properties with 38.6 emu/g saturation magnetization at room temperature. Fourier transform infrared spectroscopy and the thermal analysis study supported the formation of the nanocomposite and the enhancement of thermal stability in the resulting nanocomposite comparing with its counterpart in free state. The loading of 6-mercaptopurine (MP) in the FPEGMP-2 nanocomposite was estimated to be about 5.6% and the kinetic experimental data properly correlated with the pseudo-second order model. Also, the release of MP from the FPEGMP-2 nanocomposite shows the sustained release manner which is remarkably lower in phosphate buffered solution at pH 7.4 than pH 4.8, due to different release mechanism. The maximum percentage release of MP from the nanocomposite reached about 60% and 97% within about 92 and 74 hours when exposed to pH 7.4 and 4.8, respectively.

Enhanced Cytotoxic Activity of 6-Mercaptopurine-Loaded Solid Lipid Nanoparticles in Hepatic Cancer Treatment.

6-Mercaptopurine (6-MCP) is an antiproliferative purine analog used in acute lymphoblastic leukemia, non-Hodgkin lymphoma, and inflammatory bowel disease (Crohn's disease, ulcerative colitis). Although 6-MCP has the great therapeutic potential for cancer and immunosuppressant-related diseases, 6-MCP is not readily soluble in water, presents a high first-pass effect, short half-life (0.5-1.5 h), and implies a low bioavailability (16%). On the contrary, solid lipid nanoparticles (SLNs) are prepared from solid lipids at room temperature and body temperature. In this study, SLNs were prepared w/o/w double emulsion-solvent evaporation method using Precirol ATO5 as matrix lipid. In the emulsion stabilization, surfactant (Tween 80) and polymeric stabilizer (polyvinyl alcohol [PVA]) were used. Two group formulations using Tween 80 and PVA were compared in terms of particle size, polydispersity index, zeta potential encapsulation efficiency%, and process yield%. Differential calorimetric analysis and release properties were examined for optimum formulation, and release kinetics were calculated. According to studies, sustained release was obtained with SLNs by the Korsmayer-Peppas kinetic model. The in vitro cytotoxicity studies were performed on the hepatocarcinoma (HEP3G) cell line. According to the results, successful SLN formulations were produced, and PVA was found best stabilizer. Optimum formulation exhibited significantly higher cytotoxic effects on HEP3G than on pure 6-MCP. These results demonstrated that solid lipid nanodrug delivery systems have great potential for formulation of 6-MCP.

Investigation on absorption and release of mercaptopurine anticancer drug from modified polylactic acid as polymer carrier by molecular dynamic simulation.

The absorption and release of 6-mercaptopurine anticancer drug was investigated in biodegradable and biocompatible polymer of polylactic acid (PLA) using molecular dynamics simulation. For this purpose, the amount of mixing energy, radius of gyration, mean squared displacement and radial distribution function were computed and compared in concentrations of 5-36 wt% of 6-mercaptopurine drug. The simulation results show that increasing the concentration of the drug reduces mixing energy and PLA polymer carrier is able to carry 35.8 wt% of 6-mercaptopurine anticancer drug. Based on these results, the amount of 6-mercaptopurine release from PLA carrier 35.8 wt% of it in water environment is zero due to hydrophobic property of PLA and 6-mercaptopurine. Finally, polyethylene glycol (PEG) polymer with different percentages (10-30 wt%) was used to modify PLA carrier. The simulation results show that the rate of drug release increases by increasing the concentration of PEG polymer in the modified PLA carrier and also with increasing the percentage of drug loaded in the carrier and also the optimum weight percentage of PEG in modified PLA carrier for 35.8 wt% of drug concentration is 11 wt% and the rate of drug release is slower and equal to 4.4 molecules/ns.

A novel multi stimuli-responsive PEGylated hybrid gold/nanogels for co-delivery of doxorubicin and 6­mercaptopurine.

The clinical applications of anticancer drugs are restricted due to the incomplete delivery to the cancerous tissue and the numerous drug resistance mechanisms involved in malignant cells. In this regard, stimuli-responsive nanomaterials offer a promising prospect to deal with these concerns. In the present study, ternary responsive hybrid gold/nanogels (Au/NGs) were synthesized as a new nanoplatform to simultaneously carry two anticancer drugs, i.e., doxorubicin (DOX) and 6-mercaptopurine (MP). For this purpose, these drugs were successfully loaded (the loading capacity of 23% and 11%, respectively) into the hybrid Au/NGs by electrostatic interaction (DOX) and AuS bonds (MP). The triggered drug release ability of hybrid Au/NGs was assessed by comparing the environments of simulated physiological and tumor tissue. The incorporation of disulfide bond linkers, pH sensitive, and thermosensitive polymeric segments endowed the NGs with an excellent property in reducing acidic and hyperthermia environments, which greatly facilitates drug release in tumor cells. Intracellular tracking of DOX@MP-Au/NGs confirmed efficient accumulation and cellular uptake of developed NGs and the cytotoxicity studies showed a pronounced tumor inhibition compared with free DOX@MP. It was concluded that the new ternary-responsive NGs have great potential for co-delivery of DOX and MP and can be used in efficient cancer therapy.

Redox and pH-responsive gold nanoparticles as a new platform for simultaneous triple anti-cancer drugs targeting.

Cancer is considered to be one of the leading causes of morbidity and mortality worldwide and nanotechnology was shown to have a unique potential to enhance the therapeutic performance of anti-cancer agents. A novel dual stimuli-responsive polyethylene glycol (PEG) block copolymer was synthesized for the decoration and stabilization of gold nanoparticles (NPs) to carry multiple anti-cancer drugs, doxorubicin (DOX), methotrexate (MTX) and 6-mercaptopurine (MP). DOX, MTX and MP were successfully loaded (the loading capacity of 37%, 12%, and 49%, respectively) into the NPs by ionic interaction (DOX and MTX) and disulphide-covalent bond formation (MP) in the polymeric shell of NPs. Furthermore, the triggered drugs release ability of NPs was shown through the comparison of simulated physiological and tumor tissue environments. The enhanced efficiency of the developed NPs and their targeted performance via MTX (target ligand of folate receptors) decoration were illustrated through the various cell cytotoxicity studies such as MTT assay, DAPI staining, and flow cytometry on various cancer cell lines with different levels of folate receptors. Our proposed idea in simultaneous delivery of three cytotoxic drugs with our newly designed PEGylated gold NPs may provide promising and novel prospect in cancer therapy.

Determination of ITPase activity in erythrocyte lysates obtained for determination of TPMT activity.

The indication for the determination of both thiopurine methyltransferase (TPMT) and inosine triphosphate pyrophosphohydrolase is identical (i.e., adverse drug reactions toward mercaptopurines). Therefore, we tested whether or not our standard procedure to prepare erythrocyte lysates for measurement of TPMT activity, which includes treatment with Chelex 100 (a chelating resin), was suitable for the measurement of ITPase activity. It also was tested to see if ITPase activity differs in EDTA and Heparin anti-coagulated blood samples. We found that there was no difference between the ITPase activity in erythrocyte lysates prepared from EDTA or Heparin anti-coagulated blood. Treatment with a chelating resin or omission of magnesium from the assay procedure resulted in decreased and nearly absent ITPase activity, respectively. We conclude that untreated erythrocyte lysates obtained for determination of TPMT activity are suitable for determination of ITPase activity. However, after treatment with Chelex 100 the erythrocyte lysates become unsuitable for determination of ITPase activity.

Fabrication of biodegradable micelles with reduction-triggered release of 6-mercaptopurine profile based on disulfide-linked graft copolymer conjugate.

This research is aimed to develop a biodegradable micelle delivery system with sheddable poly (ethylene glycol) shell to achieve the reduction-triggered intracellular sustained release of 6-mercaptopurine (6-MP) and decreased toxicity. Firstly, the amino-disulfide linked poly (ethylene glycol) monomethyl ether (mPEG-SS-NH(2)) was synthesized by the amidation reaction between cystamine and active ester of mPEG and p-nitrophenyl chloroformate (p-NPC) (mPEG-NPC). And then, the five-member rings in poly (l-succinimide) (PSI) were successively opened by mPEG-SS-NH(2) and 2-(pyridyldithio)-ethylamine (PDA) to produce the graft copolymer of mPEG-SS-NH-graft-PAsp-PDA. To avoid the drug initial burst, 6-MP was covalently conjugated with mPEG-SS-NH-graft-PAsp-PDA by thoil-disulfide exchange reaction to give the resultant product mPEG-SS-NH-graft-PAsp-MP. The product was found to form spherical micelles in aqueous media because of its amphiphilic nature with average particle size of 160 nm measured by dynamic light scattering (DLS). It was found that the mPEG-SS-NH-graft-PAsp-MP micelles, though stable in phosphate buffer solution (PBS), were prone to aggregation in the presence of dithiothreitol (DTT). The in vitro drug release studies revealed the release of 6-MP were distinct from the conventional micelles whose drugs loaded by physical encapsulation. Sustained release profile of 6-MP over 85 h was found in the presence of DTT (40 mM) simulating the intracellular condition while minimal drug release was observed within 24h at the level of DTT corresponding to extracellular environment. Remarkably, the cell viability results showed there was essential decrease of cytotoxicity to HL-60 cell line compared to free 6-MP.

Does the tongue play a role in the initial development of vertical palatal shelf in hamster?

A study was undertaken to examine whether the tongue plays any role in determining the primordial development of palatal shelves in a vertical direction in mammals. Control and 6-mercaptopurine-treated embryos from Golden Syrian hamsters were examined by scanning electron microscopic and histological techniques for the spatio-temporal relationship of primordial development of the palate, tongue, and mandible. DNA synthesis, measured by 3H-thymidine incorporation, was used as an index of growth. The data indicated that in controls, vertical palate development began in the anterior half from the roof of the oronasal cavity, whereas the tongue bulges and the mandibular process developed in the posterior half of the oronasal cavity. A burst in DNA synthesis occurred in the palate and mandible, but not in the tongue. In 6-mercaptopurine-treated fetuses, although the chronological appearance of primordia of all three structures was normal, DNA synthesis was inhibited in all three structures. The recovery in DNA synthesis, albeit partial, was faster in the palate and mandible than in the tongue. On the basis of observations from the present study, along with those from other vertebrates, it is suggested that the developing tongue may not play any role in determining the direction of development of the palatal primordia.

Deoxythioguanosine triphosphate impairs HIV replication: a new mechanism for an old drug.

Inhibition of HIV-1 reverse transcriptase (RT) and HIV protease are effective mechanisms for anti-retroviral agents, and the combined use of mechanistically different medications has markedly improved the treatment of HIV infected patients. The active metabolite of mercaptopurine and thioguanine (TG), deoxythioguanosine triphosphate, was shown to be incorporated into DNA by the polymerase function of HIV-1 RT and then to abrogate RNA cleavage by HIV-1 RNaseH. Treatment of human lymphocyte cultures with thioguanine produced substantial inhibition of HIV replication (IC(50)=0.035 microM, IC(95)=15.4 microM), with minimal toxicity to host lymphocytes (<10% at 15.4 microM TG, P<0.000005). Furthermore, low concentrations of TG and zidovudine were synergistic in inhibiting HIV replication in human lymphocytes (synergy volume=19 microM(2)%), without additive cytotoxicity to host lymphocytes. Thus, thiopurines are novel anti-retroviral agents that alter the DNA-RNA substrates for HIV RNaseH, thereby abrogating early stages of HIV replication.

Changes in the oral microflora during cytotoxic chemotherapy in children being treated for acute leukemia.

Thirty-four children with diagnosed cases of acute leukemias and being treated with cytotoxic chemotherapy at St James' Hospital, Leeds, were followed for between 6 months and 1 year to determine the changes in their oral microflora. They were examined before treatment commenced and then at monthly intervals. Swabs were taken from the oral cavity to test for the presence or absence of bacteria and Candida. Saliva samples were also used to assess the levels of Streptococcus mutans in the mouth. Sensitivity tests were carried out to assess the effect of the cytotoxic agents on the oral flora. All children received prophylactic nystatin and chlorhexidine gluconate mouthrinses four times daily for the whole period of the study. There was significant difference (p < 0.0001) for counts of S. mutans at different treatment stages. Sensitivity tests showed that S. mutans was sensitive to the cytotoxic drug daunorubicin, and this drug was probably responsible for the fall in S. mutans counts. A significant difference was also found in the types of bacteria isolated between the study and reference groups, but there was no change in the composition of the flora in the study group during treatment. These bacteria were also found to mirror those cultured from routine blood samples in children with acute leukemia.