Key words doxorubicin; polybutylcyanoacrylate isobutyl; nanoparticles; multidrug resistance; reversal
Tumor cells to produce drug tolerance is the main reason for the clinical failure of chemotherapy, including multidrug resistance (multidrug resistence, MDR) tumors produce the most important mechanism of drug resistance . Chemotherapy is the preferred method for the treatment of leukemia, easy to relapse, and biological characteristics of MDR leukemia, strengthen leukemia MDR reversal study has important implications. Carrier systems (microspheres, liposomes, nanoparticles) reversal of MDR is one of the effective strategies [2 4], the experimental doxorubicin poly isobutyl cyanoacrylate nanoparticles (adriamycin loaded polyisobutylcyanoacrylate nanoparticles ADR PIBCA NP) Reversal of multidrug resistant cells strain of HL 60/ADR the MDR observe the reversal effect, to study its possible mechanism.
1 Materials and methods
1.1 drugs and reagents doxorubicin and MTT, Sigma, RPMI 1640 medium and fetal calf serum (Gibco) products, and other reagents were of analytical grade.
1.2 adriamycin nanoparticles weighed 66.5 mgα IBCA monomer (alpha cyanoacrylate the Dioxide monomer), of the electromagnetic stirring slowly added dropwise containing the adriamycin 5 mg, 5% glucose, 1% dextran 70,0.5% citric acid solution was stirred at room temperature for 6 h to complete the emulsion polymerization, 2 ml dispensing prepared, except a small amount detected after 36 h, the remaining freeze-dried sealed, stored at 4 ℃ standby. With the prepared blank nanoparticles (ie without doxorubicin nanoparticles).
1.3 nanoparticle morphology and particle size observation nanoparticles 3 bottles of bottle plus 2 ml of distilled water, wind and percussion dispersed, negatively stained with 3% phosphotungstic acid chip morphology was observed by transmission electron microscopy camera and measurement of particle size and distribution, each piece count determination of more than 200 rows of data statistical analysis.
1.4 nanoparticles encapsulating rate measurement taken two bottles, one was dissolved in DMSO: H2O (3:1), the total drug amount (WT), and the other to the the fluorescence spectrophotometric measurement of adriamycin at 488 nm fluorescence value meter taken a bottle of distilled water was added 2 ml, wind and percussion dispersed, low-temperature high-speed centrifugation (4 ° C, 000 rev / min) for 2 h after the separation of the supernatant, the same method for the determination of free drug amount (WF).
The encapsulation efficiency = WT-WFWT × 100%
1.5 cell lines and culture conditions of human leukemia cell line the HL 60/ADR in our laboratory species preservation, in ADR 0.05 μg / ml, 10% fetal calf serum RPMI1640 culture medium of stable growth. Test two weeks before switching to non-ADR RPMI1640 culture medium.
1.6 MTT method drug sensitivity test suspension of 2 × 103 well tumor cells in logarithmic growth phase 1, were seeded in 96-well plates were divided into four groups, ie doxorubicin group, adriamycin nanoparticles group, control group and blank group, the Adriamycin group and adriamycin nanoparticles group per hole plus different concentrations of ADR, the control group plus isodose solvent blank group without cells only dose solvent. Each concentration repeated four holes, set for 5% of CO2, and cultured at 37 ° C for 72 h after each hole plus 2 g · L 1 by MTT of 25 μl of 37 ° C for 4 h after the termination of culture supernatants were removed carefully added to each well 150μl DMSO, shaken for 10 minutes to fully dissolve the crystalline material. Using a microplate reader measured OD570.
Inhibition rate = (1 drug group OD / control group OD) × 100%
1.7 flow cytometry determination of the intracellular drug concentration in the tumor cells of the logarithmic growth phase suspension of 2 × 106 cells / ml, centrifuged, PBS washed once with serum-free RPMI1640 culture liquid suspension, were divided into three groups, i.e. doxorubicin group the the nanoparticle group and the control group, adriamycin group of doxorubicin and doxorubicin nanoparticles group ADR final concentration 2 μmol / L, the control group without ADR. 37 ° C and incubated for 1 h, then wash away the drugs trypan basket dyeing greater than 97% of the number of living cells. Filtered cells measured by flow cytometry the intracellular adriamycin relative fluorescence intensity (excitation wavelength 488 nm, emission wavelength 575 nm), to the representatives of the relative fluorescence intensity of doxorubicin, doxorubicin concentration.
1.8 statistical methods were compared t test.
2.1 morphology and particle size observed composite transmission electron microscope was spherical, granular, no adhesions, the average particle size of 98.5 ± 3.67 nm (figure omitted).
2.2 doxorubicin encapsulation efficiency was 95%.
2.3 MTT illicit drug sensitivity test blank nanoparticles at high concentrations (100 μg / ml) significantly inhibited the proliferation of tumor cells at low concentrations (10 μg / ml) had no inhibitory effect on tumor cell proliferation, indicating that the cytotoxicity of nanomaterials The role of the MTT assay negligible. Adriamycin nanoparticles and doxorubicin HL 60 cell cytotoxicity; the HL 60/ADR in any concentration point, adriamycin nanoparticles compared to doxorubicin toxicity significantly enhanced its reversal fold was IC50 (ADR) / IC50 (ADR PIBCA NP) = 2.63 times.
0.05）；而在HL60/ADR细胞内，则阿霉素纳米粒">2.4 intracellular doxorubicin concentrations of doxorubicin nanoparticles group and the doxorubicin group, in HL-60 cells ADR concentrations were similar (P> 0.05); within in HL 60/ADR cells, doxorubicin nanoparticles the group ADR concentration was significantly higher than doxorubicin group, about 1.48 times the doxorubicin group.
Nanoparticle as nanoscale carrier system, can effectively protect the drugs, controlled release and targeted release characteristics, which significantly improve the efficacy, reduced toxicity and become one of the hot spots of the Pharmaceutical Research. Nanoparticles at this stage in the human body to absorb almost empty and pharmacodynamic study the mechanism of action of nanoparticles also more controversial, so further research is necessary. Due to the higher requirements of nanoparticles preparation conditions, have not yet become a commercial formulation, and other factors, is usually difficult to obtain in the country, thus affecting the launching of nanotechnology research.
Nanoparticles methods include double emulsion method, adsorption, solvent the non-solvent, emulsion frozen melting method . The nano-material selection is divided into the biodegradable type, such as polylactic acid, polyglycolic acid, polylactide ester, polycyanoacrylates alkyl acrylate, etc., and non-biodegradable, such as poly (methyl methacrylate), polystyrene, polyamide, etc.. As the biodegradable material in degradation at the same time accompanied by drug release, and the polymer materials in vivo hydrolysis enzyme eventually produce water and carbon dioxide, its application than the non-biodegradable increasingly widespread. Encapsulant for peptide and protein-based chemotherapy drugs. The laboratory reference foreign literature, the assistance of the College of Pharmacy, emulsion polymerization synthesis the adriamycin poly isobutyl cyanoacrylate nanoparticles (adriamycin loaded, polyisobutylcyanoacrylate nanoparticles ADR PIBCA NP). The emulsion method using surface active agent so that the polymer monomer emulsion into droplets of nano-size, and then the monomer is polymerized into the polymer (in this experiment is the use of hydroxyl ions so that polymerization). Detect nanoparticles was spherical particles, the average particle size below 100nm, doxorubicin encapsulation efficiency was 95%, as described in foreign literature quite fit the experimental requirements, provide good conditions for the further study of nanoparticles.
Multi-drug resistant (MDR) tumor cells to anticancer drugs is the main reason for the failure of cancer chemotherapy, and the reversal of MDR is one of the hotspots of cancer research. Chemical modification the reversal strategies include drugs , chemosensitizer applications . Soma CE  and Barraud L  studies show wrapped doxorubicin poly isobutyl cyanoacrylate nanoparticles can effectively reversing tumor cells MDR mechanisms to avoid the multidrug resistance glycoprotein (P gP) role. Laurand A  found that poly isobutyl cyanoacrylate nanoparticles can reversal of MDR leukemia cell line K562 improve intracellular drug concentration and its reversal of one of the mechanisms. Barraud L  for further study that, PIBCA adriamycin nanoparticles reversal of MDR by changing some of the characteristics of the cell membrane rather than a direct effect on the P gP.
The nanoparticles can reverse tumor cell MDR, there are many articles reported in the literature, but are of P gP mediated MDR, no reverse against the MRP test report. Numerous studies show that the MRP either alone mediated mediated leukemia MDR also participate. Our results show that: ADR of PIBCA NP groups on the HL 60/ADR cytotoxicity is 2.63 times the ADR group ADR of PIBCA NP groups in HL 60/ADR intracellular ADR concentration ADR group 1.48 times. The ADR PIBCA NP can effectively reverse MRP-mediated MDR significantly increased intracellular ADR concentration of its mechanism. Increase in intracellular drug, Mo Y  that enter cells by endocytosis avoid film Pump efflux effect, Chavanpatil MD  analysis for a large number of drugs diffuse into the cell saturated of P gP of efflux . Despite the existence of other mechanisms yet to be further studied, but the results of our research is to determine: HL 60 cells on the sensitivity of ADR and ADR PIBCA NP,, ADR PIBCA NP the HL 60/ADR cytotoxic large in ADR. Studies have shown that MRP and P gP some similarities, but the former can also affect the GSH / GST system, and promote drug redistribution within the cells leave targets , so do not rule out other mechanisms are involved in the possibility of .
Nanoparticles nanoscale as a drug carrier system, because of its good controllability of the release of pharmacokinetic parameters may be changed in a certain extent can effectively penetrate biological barriers has the significance of in-depth study, and its wide range of material selection variety, preparation methods gave further study the possibility.
Although nanoparticles in injection, oral, topical, there are still many problems to be solved, but its broad application prospects are obvious. With the development of the pharmaceutical industry is expected to continue to develop new, safe, and clinical application prospects carrier material; carrier mechanism, the carrier material and drug interactions way is bound to get more in-depth and extensive research.