| Background and ObiectiveIn the 70y 20th, with the extensive application of modern scientific instruments, nanomaterials and nanotechnology attracted peoples’ attention and focus, and gradually be used in pharmaceutical field, resulting of nano-delivery systems. In the 90y 20th, it began to become a pharmacy one of the hot areas of research. The so-called nano-drug delivery system refers to the drugs and medicinal materials, together with the formation of particle size of 1-1000nm of the nano-scale drug delivery systems, in recent years it has been very actively in the field of pharmaceutical of a series of new drug delivery systems collectively referred to as ultra small. Nanoparticles is a natural or synthetic polymer material particle size which was between 1-1000 nm solid colloidal particles, including nanospheres and nanocapsules. Active component (drug, biologically active materials, etc.) dissolved, encapsulated in the internal particles, or adsorbed, attached to the particle surface. NP has numbers of advantages:a non-biological materials with none of immunogenicity and cell toxicity which has a higher gene transfection efficiency, availability of long-term stable expression of target genes; it can protect the drugs or the target gene from plasma or tissue cells of the body damaged a variety of enzymes and complement. NP through the surface or through special materials targeting to tumor, achieving better results which become cancer therapy research focus.Leukemia, a large kinds of hematopoietic malignancies, is a serious threat to human health, the current treatment methods include chemotherapy, hematopoietic stem cell transplantation and molecular targeted therapy. Chemotherapy, only achieving temporary relief, and the side effects is obvious. Allogeneic stem cell transplantation is limited due to donor, graft versus host disease and transplant-related mortality and economic conditions and other factors severely; fewer than half of patients may accept such treatment. The application of molecular targeted therapy is also subject to certain limitations. This provides us the search for new targeted therapy drugs issue.Development of natural products in recent years is the development of new anti-leukemia drug ideas. Artemisia from the Compositae extracts in China, containing peroxide groups within (-COOC-) with the structure of antimalarial sesquiterpene drug. To improve the water-soluble, semisynthetic of artemether, srteether, dihydroartemisinin and artesunate and other derivatives, appeared one after another. Recent studies have found that artemisinin and its derivatives have strong inhibitory effects on various tumor cells. Artesunate has inhibited 55 kinds of human tumor cell lines, which is the most sensitive for the leukemia and colon cancer cells, moderately sensitive to melanoma, breast cancer, ovarian cancer, prostate cancer, renal cancer and other central nervous system tumors, the the worst sensitivity is non-small cell lung cancer, the U.S. National Cancer Institute has incorporated anti-cancer drug screening and research programs in the anti-cancer activity.Preliminary studies by our groups confirmed that the anti-tumor effect of Art mainly in tumor cells of the rich in the iron-catalyzed peroxidation within the Art of the fracture group generated carbon-centered radicals, triggering caspase apoptosis pathway.Over-expression on tumor cell surface express transferrin receptor 1 (CD71), Art may also reduce the expression of TfRl on leukemia K562 cells by TfRl-Tf way to reduce their intake of iron, by reducing the second divalent metal ion transporter (DMT1) expression of the cell, deducting from the endosome transport to the cytoplasm of the Fe2+, mitochondria can use iron also decreased, by reducing the membrane iron transporter protein 1 (FPN1) mRNA expression of the cell to reduce the outflow of iron, thus inhibiting cell proliferation and induce apoptosis.Research has shown that hollo-transferrin (hollo-Tf) on covalent binding to Art greatly enhanced inhibiting the growth of tumor cells than alone Art.The results told us Art combinating of Tf had synergistic effect, which provides a scientific idea for further develop new formulations of anti-tumor Art. focusing on reducing drug toxicity, prolonging its half-life, increasing bioavailability, enhancing anti-tumor efficacy, reducing drug resistance, the Art nanoparticles is made.General NP get into the circulatory system, but was quickly cleared by he system of mononuclear phagocytes (MPS) macrophages and polymorphic nuclear leukocyte, it is difficult to reach the target tissue and target cell, the target tissue deposition less, in order to avoid clearing and improve the drug deposition in target tissues, to extend its circulation time in vivo, increased blood-brain barrier penetration of drugs. In the preparation process, the use of certain hydrophilic and flexible of polymer on the surface modified nanoparticles to enhance the stability of ordinary NP, such as described in this article using polyethylene glycol (PEG) and poloxamer was modified. Poly lactic acid-glycolic acid (PLGA) by the U.S. FDA approved for human use of biodegradable polymer, with methoxy polyethylene glycol (mPEG) modified (mPEG-PLGA) to make up carrier, as the surface of Poloxamer, made of a new type of long-circulating nanoparticles, also known as invisible nanoparticles, or "space stability" nanoparticles.In order to prepare the NP with active targeting, the tumor cell surface binding Tf, Tf-TfR1 specific combined to the tumor cell surface, so that drugs can be accurately sent to the target, to achieve tumor tissue (cells), reducing dose and toxicity. Tf are kinds of glycoprotein involved in body iron transport, transferrin and transferrin receptor-mediated endocytosis of biological cells is one of the most characteristic of the transfer process, therefore transferrin become a natural target ligand favored. In this study, first part formed of Tf-mPEG-PLGA-Art-NPs (referred to Art-NPs), next part study of its inhibiting K562 cells, the last part studied its metabolism and distribution. Methods1.Modified-spontaneous emulsion solvent diffusion method(modified-SESD method) were prepared for Art-mPEG-PLGA-NPs, using Marshall’s method for fluorescein isothiocyanate (fluorescein isothiocyanate, FITC) labeled Tf, they soluted with the ratio of 1:1,4°under slow, reacting products of high-speed centrifuge 30min, sedimentation shall Tf-mPEG-PLGA-Art-NPs (Art-NPs).2. The shape of the nanoparticles was observed by SEM. The mean diameter and the size distribution of nanoparticles were determined by laser light scattering. The drug loading efficiency, encapsulation rate and releasing behavior of Art-NPs in vitro were examined by HPLC.3. MTT assay at different concentrations and different time points detected Art-NPs on human leukemia K562 cells in vitro growth inhibition.The apoptosis was analyzed by invert microscope from Hoechst33342 staining. The phagocytosis ability is stained for Wright Giemsa staining.4. Each experimental group over time were treated of two kinds of NPs (mPEG-PLGA-Art-NPs and Tf-mPEG-PLGA-Art-NPs). HPLC detected plasma concentrations at 15min,25min,30min,40min,60min,120min,180min,240min.On the while, we observed contribution in the liver, spleen, bone marrow at 15min, 40min,60min.5. The experimental data were descripted of mean±standard deviation ((?)±s). The experimental data between the rates of cell proliferation were treated by repeated measures ANOVA and One-ANOVA. Pharmacokinetic parameters were deal with Winonlin software. Different concentration data at different times of NPs were deal with factor analysis and One-ANOVA. Application of SPSS 13.0 software for statistical analysis. P<0.05 is stands for statistics difference.Results1. Art-NPs of the form, distribution and characterization of physical and chemical properties.Art-NPs in the naked eye under a transparent pale blue opalescent solution, there are no visible precipitation exists, place 3 months was not observed with phase separation or flocculation occurs. The nano-suspension drops a small amount added to a clean glass slide, light microscope observation of 100 times zoom lens, a large blue dot scattered in small particles, spherical or spherical, evenly distributed, no adhesion phenomena. Observed under transmission electron microscope for the spherical entity particles, the surface smooth, with an average size range of (156.7±1.01)nm, zeta potentials ranging from-(26.23±1.86) mV, the average drug loading (14.5±0.2)%, the average entrapment efficiency to (86.5±0.5)%. F/P=1.8. The NPs combination rate of Tf was average 13.5%.2. Art-NPs’proliferation on the k562 cell and release conditions in vitroFive kinds of tumor cell lines (HL-60、Molt-4、K562、Jurkat、Rajj) express CD71(+) percentage were average 98.85%、95.76%,90.84%,84.16%,72.69%. MTT assay results showed that the creation Art-Nps with concentration of 12.5μg/ml、25μg/ml、50μg/ml、100μg/ml as the experimental concentration, different concentrations (F=407.469, P=0.000) at different time points (F=1301.003, P =0.000) of the Art-Nps group could inhibit K562 cell proliferation, and both have a synergistic effect. With the processing time extending and the dose increasing, cell proliferation inhibition rate increased gradually, showing a clear time-dose dependence from the experimental group 12.5μg/ml to 100μg/ml after 24h、48h、72h (P<0.01).Compared with 50μg/ml and 100μg/ml, they have no statistics difference. 100μg/ml Art-NPs deal with K562 cells at 72 hours, inhibition rate was 70.4%(>67.1 vs control groups), which is sustained-release effect. Art-NPs of the cells resulted in significantly higher apoptosis than blank groups. Wright Giemsa staining found more acidophilia particles were swallowed by K562 cells and macrophage in abdominal cavity of mice after 48h vs 24h.3. In vitro release of Art-NPsIn vitro release of the laws in line with Higuchi equation:Q=4. 11t1/2+27.05, R2 =0.983. Releasing of 5 days were up to 70%.4. Art-NPs’pharmacokinetic characteristics Blood concentration-time curves of Tf-mPEG-PLGA-Art-NPs and mPEG-PLGA-Art-NPs appeared two peaks.The results of two kinds of nano-formulations and control group were significantly different, AUC (area under the concentration-time curve) and AUMC (area under the first moment of the concentration-time curve) of PEG group and Tf group were significantly greater than the Art group (3.0 times/9.04 times,2.03 times/8.43 times). And Cl (clearance rate) was significantly lower than the control group (P<0.01). The two experimental groups of T1/2 (elimination half-life) were 126.63min and 172.76min, were significantly higher (P<0.01). The MRT (mean retention time) of two experimental groups were also significantly higher, Vss (steady-state apparent volume of distribution) of PEG Group had no significant difference compared with the control group, but Vss of Tf group was significantly higher than Art Group.5. Art-NPs’ contribution in vivoDistribution of Art in vivo of mice (1)at 15min after administration:blood (28.40±0.10 vs.58.40±0.44, P=0.000)、liver(11.54±0.07 vs.4.56±0.06, P=0.001)、spleen (48.56±0.85 vs.9.97±0.02, P=0.000) increased, bone marrow (11.52±0.10 vs.27.07±0.29, P=0.000) decreased of PEG group VS control group.blood (79.95±6.65 vs.58.40±0.44, P=0.000)、liver (5.13±0.07 vs.4.56±0.06, P=0.001)、spleen (12.66±0.36 vs.9.97±0.02, P=0.001) increased, bone marrow (2.27±0.07 vs. 27.07±0.29, P=0.000) decreased of Tf group VS control group.(2)At 40min after administration:liver (50.60±0.53 vs.0.90±0.10, P=0.000)、spleen(36.70±0.27 vs. 11.80±0.27, P=0.000) increased of PEG group, blood (71.70±0.80 vs.45.70±0.61, P=0.000)、liver (6.30±0.26 vs.0.90±0.10, P=0.000) increased, spleen(4.33±0.61 vs 11.80±0.27,P=0.000)、bone marrow(17.60±1.15 vs 41.6±1.41, P=0.000) decreased of Tf group.(3)At 60min after administration:blood (19.00±0.12 vs.19.00±0.12, P=0.000)、bone marrow (15.30±0.03 vs.29.59±0.65, P=0.000) decrease, liver (7.18±0.05 vs.4.94±0.03, P=0.000)、spleen (58.52±0.12 vs.14.46±0.66, P=0.000) increased of PEG group; blood (52.08±0.55 vs.49.97±0.66, P=0.002)、 liver (7.34±0.0.03 vs.4.94±0.03, P=0.000)、spleen (18.09±0.25 vs.14.46±0.66, P=0.000)increased of Tf group, bone marrow(22.48±0.55 vs.29.59±0.65, P=0.000) decreased of Tf group.Art Contribution of different organs and tissue:(1) blood:three groups at different time were interaction (F=72.622, P=0.000), group between groups have significant differences (F=935.619, P=0.000), time between time have significant difference between the (F=557.151, P=0.000). (2) liver:interaction between the three groups (F=3410139, P=0.000), group between groups were significantly different (F=3362905, P=0.000), time between time were significantly different (F =4382466, P=0.000). (3) spleen:interaction between the three groups (F= 8617.393, P=0.000), group between groups were significantly different (F= 63409.888, P=0.000), time between time were significantly different (F= 63409.888, P=0.000). (4) bone marrow:interaction between the three groups (F= 7768.881, P=0.000), group between groups were significantly different (F= 27298.373, P=0.000), time between time were significantly different between the (F =60274.729, P=0.000).Conclusions1. Art-NPs with the size of a small, high drug loading and entrapment efficiency of the characteristics of in vitro release showed a good slow release, indicating preparation method workable and practical for the nano-emulsion preparation of artesunate to provide an important reference value.2. In vitro experiments proved Art-NPs alone both inhibit human leukemia K562 cell proliferation and apoptosis, the effect was time-dose dependent, with time and dose increases, a significant increase in tumor suppressor role. Cells 72h after the Art-NPs inhibition rate of more than Art alone, indicating Art-NPs can extend the role of drugs on leukemic cells the time, with sustained-release effect in vivo.3. Art-mPEG-PLGA-NP as a release agent, the drug release increased gradually from 1-5d,50h later, over time, then released into a relatively gentle process. The results show that, Art-mPEG-PLGA-NP with time, through continuous release of artesunate, and maintained at a high concentration, thus prolonging the effect of artesunate on the active time of tumor cells.4. Artesunate drug metabolism the body of mice to follow after the doctrine of compartment model, blood and bone marrow concentration having a relatively high value. Nanoparticles go into body, a large number of sheets swallowing by the macrophage phagocytic system, as time goes by, the liver concentration decreased, but still remain a lot of the spleen nanoparticles. Art-NPs maintain the highest blood concentration in the body, the bone marrow followed, blood and bone marrow concentration was still at a relatively high value. This shows that the new nano dosage form has blood and bone marrow targeting, which is the treatment of leukemia and other blood malignancies provide a basis for treatment has important clinical value. |
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