| Cancer is the leading cause of human being death in the world. Based on the World Health Organization (WHO) statistics, there are about.7.1 million cancer deaths and 8.7 million new cancer cases, which are still increasing annually. Lung cancer was the most commonly diagnosed cancer as well as the leading cause of cancer death in males in 2008 globally. Non small cell lung cancer (NSCLC) accounts for 87% of all cases, with a dismal 5-year survival rate of 15%. Almost two thirds (70%) of patients present locally advanced disease or metastatic disease at the time of diagnosis,the primary treatment of choice for which is chemotherapy.Docetaxel (DT) is extracted from the semi-synthetics in European yew tree,which is the most promising chemotherapeutic agents for the treatment of non-small-cell lung cancer (NSCLC). Docetaxel plays antitumor effect via cell tubulin binding, inducing cell apoptosis and cell cycle arrestment at the molecular level. But docetaxel dosing and efficacy are limited by its undesirable side effects, including febrile neutropenia, myelosuppression allergic reactions, hair loss, fluid retention, and skin reactions. Docetaxel is insoluble preparations need polysorbate 80 (Tween 80)involved in its clinical formulation (Taxotere) as a solvent to help dissolve, but Tween 80 can cause hemolytic and allergic reactions. In order to improve the tumor-specificity, therapeutic efficacy of DT and reduce the toxicity of health tissue,nanoparticular drug delivery systmes such as nanoparticles, liposomes, micelles or dendrimers etc. have been widely investigated. Polyethylene glycol-distearoylphosphatidylethanolamine conjugate (PEG-DSPE) is a nano carrier for drug delivery, which is amphiphilic polymer micelles formed by conjugation of soluble copolymer with lipid. It has the lowest level of protein or cellular absorption,non-immunogenicity and toxicity. DT conjugated with PEG-DSPE exhibit many advantages as follows: (?) Effectively improve the low solubility of docetaxel to avoid side effects caused by the Tween 80. (?) It can evade the reticuloendothelial system(RES) of the body non-selective removal.(?) It can extend the half-life of the body and selective accumulation in solid tumors through the enhance permeability and retention (EPR) effect. (?)High stability suitable for filter sterilization and intravenous administration.At present, a variety of new target ligand or antibody modification of polymeric micelles are developed to enhance docetaxel tumor targeting and therapeutic efficiency. These specific "vector" molecules showing selective combination with receptors or antigens overexpressed on the tumor cells or tumor vascular endothelial cell surface, enhanced selective absorption and improve the activity of the drug.Integrin family is a group of glycoprotein expressing cell surface, such as?5?1,???3,?4?1 etc.which involved in cell adhesion receptors. It is over-expressed in activation of endothelial cells, tumor neovascultature endothelial cells and malignant tumor cells. It is responsible for anchoring these cells to the extracellular matrix (ECM). The arginine-glycine-aspartic acid (RGD) peptide sequence is a specific ligand of Integrin, and can recognize cell surface specific Integrin of various types. It was widely used in oncolytic viral vectors,molecular targeting drug delivery carriers, cancer therapy targeting drugs, as well as the probe, radioactive tracers for tumor imaging. As the RGD characteristics of this specific targeting of Integrin receptors, RGD-based drug delivery system is now one of the most developments targeted cancer therapy approaches.In this regard, we prepared targeting polymeric micelles DT-RGD loaded with DT using DSPE-PEG and modified them with RGD peptide on the surface. We hypothesized that the anti-cancer efficacy of DT may be further improved when we developed polymeric micelles using PEG-DSPE which was biocompatible and biodegradable amphiphilic block copolymer. It could prolong the circulation time to increase DT accumulation in tumor by the EPR effect. The polymeric micelles DT-RGD that could be functionalized with RGD peptide for tumor-targeted delivery and high cytotoxic and uptake effciency by targeting A549 cells. We estimated the novel therapeutic approach efficacy of DT-RGD in vitro and in vivo and investigated the possible related mechanism in human non-small-cell A549 lung cancer cell in order to achieve the hypothesis.Main methods and result of this research:1. Preparation of DT-RGD by the thin film methodThe activated DSPE-PEG (NHS-PEG-DSPE) was used to conjugate cRGD to PEG-DSPE. The mixture was dialyzed against water for 2 days to remove all impurities and then lyophilized. Docetaxel, PEG-DSPE, and cRGD -PEG-DSPE were co-dissolved in acetonitrile at proper proportion. After stirring for 1 h, the acetonitrile was removed under vacuum at 25?. The film was then heated to 60 ? and hydrated with 5 mL of saline. The micelles solution was then filter through 0.22 ?m microporous membrane filter to remove the docetaxel precipitates.2. FT-IR spectra analysis and nulcear magnetic resonance (1HNMR) analysis of DT-RGDAn activated DSPE-PEG (DSPE-PEG-NHS) was used to conjugated cRGD and NHS group of the lipid , which acts as the linking agent.FT-IR spectra analysis and 1H NMR were used to confirm the conjugation of cRGD to micelles. The appearance of phenyl protons of cRGD at 7.4 ppm verified successful conjugation of cRGD the surface of micelles.3. Particle size and zeta potential measurement of DT-RGD with dynamic light scattering methodThe particle size and distribution of the micelles was determined by dynamic light scattering (DLS) analysis using Malvern Zetaszer Nano ZS (Malvern Instrument Ltd, Malvern, UK) with 633nm laser at 25? and at a scattering angle of 90° at a polymer concentration of 1 mg/mL. The mean particle sizes of DT-RGD were 23.0±2.1 nm,which was larger than that of nontargeted micelles loaded with docetaxel (DT-DSPE) was 17.6±1.7 nm. It could be attributed to the expansion of micellar corona after cRGD molecules stretched out of the surface of micelles. The zeta potential of micelles with or without cRGD was -30 to -35 mV, which means the micelles could maintain their stability of dispersion.4. Drug Entrapment efficiency and Drug content with high-performance liquid chromatography (HPLC) method and high-speed centrifuge methodTotal amount of docetaxel and the encapsulated in the micelles were determined using high-performance liquid chromatography (HPLC) analysis. 0.5 mL docetaxel-loaded micellar solution in water was diluted with 3 times acetonitrile to disrupt the micellar structure. Total amount of docetaxel were measured using by high-performance liquid chromatography (HPLC). 0.5 mL docetaxel-loaded micellar solution was centrifugated for 2 h at 50000rpm. The drug content in the supernatant after centrifugation was measured by HPLC to determine the docetaxel unencapsulated in the micelles. Drug Entrapment efficiency and Drug content of DT-RGD was 97,37505%, 8.423172.5. DT-RGD observation with transmission electron microscopy(TEM)A drop of cRGD conjugated micelles suspension was placed on a TEM copper grid coated with carbon film and dried at room temperature. Morphological observation was performed using a Hitachi H-7650 transmission electron microscope(Japan) at 80 kV. The particles showed spherical shapes with uniform particle size.6. Release characteristics measurement with in vitro release studiesIn vitro release of docetaxel from DT-RGD micelles was performed as follows: 1 mL of 1 mg/mL DT-RGD micelles was incubated in a dialysis bag (MWCO 10KD)and immersed in 40 mL PBS containing 0.1% (v/v) Tween 80 at 37? with a shaking rate of 100 rpm. Sample (0.5 mL each) was withdrawn from the medium at designated time intervals and same volume of fresh medium was added. Each sample was centrifuged at the speed of 14,000 rpm for 30 min. The amount of docetaxel in each sample was analyzed by HPLC method. The docetaxel in the DT-RGD micelles was continuously released over 24h.7. Cell viability using MTT assayA549 cells were seeded in 96 multi-well plates at 8000cells/well in 200 ?L of medium containing 10% FBS. The cells were allowed to attach for 24 h, the medium was changed with fresh medium supplemented. MTT (3-(4,5-dimethylthiazol -2-yl)-2,5 -diphenyl tetrazoniumbromide) solution was added to the cultures at a terminal concentration of 1 mg/mL and incubated for another 4 h at 37 ?. The supernatant was carefully aspirated, and 200 ?l dimethylsulfoxide (DMSO) was added to dissolve formazan. The 96 well microplates was then transferred to the microplate reader for measuring the absorption at 570 nm. The absorbance was used as a measurement of cell viability. All experiments were repeated three times. The 50% inhibiting concentration (IC50) of DT,DANPS and PEG-DANPs group was 7.54±0.17?g/mL,5.2314±0.13?g/mL,4.4209±0.15?g/mL respectively. DT-DSPE and DT-RGD group showed significantly enhancement of the cytotoxic effect compare with DT group(P<0.01).8. Morphological observationCertain amount of A549 cells were seeded in 12-well plates and cultured 48h before treatment as follows: group A: Control(RPMI-1640 medium only),group B:free DT, group C:DT-DSPE, group D: DT-RGD at dosage of 5?M.After 48h incubation, cells were washed with PBS twice and stained by hematoxylin-eosin(HE)according to the Hematoxylin and Eosin Staining Kit(Beyotime Institute of Biotechnology,Shanghai China) manufacture's protocol.The stained cell were observed and photographs were taken under the light microscope. Compare with Control, free DT or DT-DSPE group, there was more pyknosis, karyokinesis and a larger cell-free zone in the cells treated with the equivalent DT-RGD.9. Apoptosis observation with fluorescence microscopyThe apoptosis was detected by assessment of morphology staining with AnnexinV-FITC/PI Apoptosis Detection kit (Beyotime Institute of Biotechnology,Shanghai China). Briefly, about 2.0×104 A549 cells were seeded into 12-well plate and cultured 48h before experiment. Then the cells were washed by PBS twice and cultured with group A: Control (RPMI-1640 medium only), group B: free DT, group C:DT-DSPE, group D: DT-RGD at pre-determined concentration. After 48h incubation,cells were stained using the 20?L fluorescein isothiocyanate(FITC) and 20?L propidium iodide (PI) according to the manufacture's protocol .The stained cells were observed and photographs were taken under the fluorescence microscopy. DT-RGD group can be observed more late apoptosis-based changes.10. Apoptosis determinations and Cell cycle analysis with flow cytometryAnnexin V-FITC/PI kit was used to measure the percentage of apoptosis induced by DT-RGD,DT-DSPE and free DT at dosage of 5?M.After treated for 48h,cells were havested (1×106 cells) and washed with PBS at 4? and the resuspended in 500?L binding buffer containing 5?L of AnnexinV-FITC and 5?L of propidium iodide(PI).After incubated away from light for 30min at room temperature, stained cells were analyzed by flow cytometry at 488nm excitation wavelength. The DT-RGD group may induce more later periods cell apoptosis compared to other two control groups(p<0.01).Cell cycle analysis was assessed by flow cytometry. The exponential-phase A549 cells incubated with DT-RGD, DT-DSPE and free DT at dosage of 5?M for 48h were havested and washed with PBS, respectively. After fixed in 70% ethanol for 12h at 4?, these cells were washed twice and resuspended in 100?L Rnase solution(100?g/mL) and incubated for 30 min at 370C .Then,400?L of PI was added followed by another 30min incubation at 4? away from light. Fluorescence was quantified on a flow cytometry at 488nm excitation wavelength, and the percentage of cells in each phase was calculated using Modfit software. DT-RGD group than the control group was significantly inhibited cell proliferation (p<0.01). DT-RGD group can be observed a high proportion G2/M cell cycle block compare with the other two treatment group (p<0.05).11. Bcl-2?NFkBp65?VEGF?MMP-9 expression with RNA extraction and real-time polymerase chain reaction(real time-PCR)The A549 cells were cultured as above and incubated with free DT, DT-DSPE and DT-RGD for 48h in RPMI-1640 with 10% FBS. Total RNA was prepared using the Trizol reagent according to the manufacturer's specifications and quantified spectrophotometrically by measuring absorbance at 260nm and 280nm. The products were subject with Line-Gene fluorescent quantitation PCR detection system. The Bcl-2?NFkBp65?VEGF?MMP-9 mRNA expression of DT-RGD group decreased significantly compare with the other three goups (p<0.01).12. Motility and migration ability with Wound healing assay and Cell invasion assayWound-healing method was used to assess the alterations of A549 cell motility and migration ability after the treatment.Briefly, 2.0×104 cells were seeded into a 12-well plate and allowed adhere to forming monolayer cell before the experiment.An artificial "wound" was carefully created using a plastic micropipette tips top to scratch on the monolayer cell. Floating and loosely attached cells were carefully removed by PBS wash and cells were cultured with DT-RGD, DT-DSPE and free DT at dosage of 5?M respectively. The time-dependence of wound closure was compared to the four groups. Photographs were taken under microscopy (OLYMPUS, CKX41)immediately after wound incision and every 12h. The area of each wounding area was calculated by Image J software. The significant inhibition of A549 cells healing recovery of DT-RGD group was detected compared with the other three group(p<0.01).A549 cells were cultured with DT-RGD, DT-DSPE and free DT at dosage of 5?M for 48h. Afterwards,the cells were removed by trypsinization and tested for in vitro invasiveness by the Corning Incorporated Transwell 3422 (6.5mm diameter,8-?m pore size polycarbonate membrane). The above treated 1×105 cells were seeded to the upper part of chamber in 200?l of serum-free RPMI1640 medium. The bottom chamber contained standard RPMI1640 medium with 10% FBS. After the chamber was incubated at 37? for 12h, the cells that had invaded to the lower surface of the membrane were fixed with formaldehyde and stained with hematoxylin solution. Cell numbers in randomLy selected five fields each well were counted under a light microscope at x400 magnification. The significant inhibition of A549 cells cell-penetrating ability of DT-RGD group was detected compared with the other three group (P<0.01)13. In vivo tumor growth inhibition study with A549 xenograft modelFive to six weeks old male BALB/c nude mice were divided into four groups randomLy experimental and control groups of six animals each. When the mice weighed 16?8g, each of 5×106 A549 cells was inoculated subcutaneously in the right flank. When the xenografts of human non-small-cell lung carcinoma size reached 80?100mm3 in volume, a certain amount of DT-RGD,DT-DSPE and free DT were respectively dispersed in saline, then were administered intravenously via the caudal vein at a dose of 10mg/kg. All the animals were treated three times at a 3-day interval(Days 13,16,and 19). The tumor inhibition activity was assessed with tumor volume at a 3-day interval. The body weight was measured simultaneously. The toxic side-effect of DT-RGD group was similar with DT-DSPE group, significantly reduce compare with Control and DT group (p<0.01).Conclusions of this research:1. The preparation of cRGD peptide modified PEGylated docetaxel targeting polymeric micelles (DT-RGD) was successfully constructed.2. In vitro,DT-RGD showed obvious cytotoxic and facilitated more late apoptosis of A549 cells compared with DT-DSPE and free DT. A549 cell line strongly induced apoptosis by the down-expression of Bcl-2 and NFkB mRNA with DT-RGD.3. A549 cell line survival rate and mobility was strongly inhibited and the expression of VEGF?MMP-9 mRNA decreased obviously following culture of A549 cells with DT-RGD.4. It can be demonstrated DT-RGD can more effectively inhibit tumor growth and less body weight loss in A549 xenograft model... |
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