| Breast cancer is the most common malignancy in women, is the main cause of death. There are 1.38 million new confirmed cases in 2008 alone worldwide, 458,400 patients died of breast cancer.Despite health care knowledge gradually increase, the more extensive scope of the screening program, more and more advanced treatment strategies, about 20-30% of the patients eventually relapse and 5-7% of patients had distant metastases . So far, the most important means of breast cancer treatment still rely on traditional methods such as surgery, chemotherapy and radiotherapy.Docetaxel is the most effective chemical drugs in the treatment of breast cancer in terms of early and progress of breast cancer, and taxane-based chemotherapy is the standard treatment program. Docetaxel was separated at first in the precursor form existence material from the European yew needle.It is very strong hydrophobic, when vein for medicine needs the solvent, the problem of water insolubility has limited its clinical practice in very long period of time. Docetaxel is generally Polysorbate 80(Tween 80) and ethanol diluent to solubilization, and then normal saline or 5%glucose solution dissolved intravenous administration. Cosolvent has some biological and pharmacokinetic activity, the body will produce some adverse reactions. The most common side effects in the Tween80 are acute allergic reactions and fluid retention.There are 40% of patients with relatively mild reaction, such as facial flushing, rapid heart beat, etc., about 1.5-3% of patients with life-threatening breathing difficulties,severe side effects of hypotension. Other toxicities related to Tween 80 are neutropenia thrombocytopenia and peripheral neuropathy. Moreover, Tween 80 leaches plasticizers from polyvinyl chloride materials, which can cause serious or even fatal allergic reaction. Docetaxel administration require special injection equipment ,prolonged the injection time, injection of steroidal and anti-histamine drugs prior to administration to prevent an allergic reaction. High doses of docetaxel does not improve efficacy, but will have a greater toxicity to normal tissue. How to avoid the use of these solvents, avoid unnecessary side effects, and more efficient delivery of high doses of drugs to the tumor site, high efficiency and low toxicity anti-tumor effect, prompted the birth of albumin nanoparticles technology.Albumin is a plasma protein of molecular weight of 66kD, is a variety of multi-function of targeted drug delivery carrier. Albumin is the maximum concentrations of plasma proteins in the body (35-50g/L plasma proteins), and natural carrier of hydrophobic molecules, has the characteristic of non-Covalent binding of hydrophobic molecules. The albumin dissolves in the pH4-9 scope in the water and the ethyl alcohol, may heat at 60 ℃ 10 up to 10 hours. The albumin does not have the immunogenic, has the very long half-life (average 19 days), so is the most potential drug delivery carrier. Albumin nanoparticles is the ideal carrier for lipophilic drugs,the combination of docetaxel with human serum albumin under high pressure into the albumin drug nanoparticles, particle size is generally in the 100-200nm. Docetaxel and protein to form a stable physical properties of particles, average particle size of about 130 nm colloidal suspensions, the particles have a hydrophobic core with the hydrophobic drug molecules, surrounded by a hydrophilic shell, undenatured negatively charged amino acids in the serum albumin. Albumin nanoparticles technology can transport hydrophobic drugs into the body, do not need to have the potential toxicity of the solvent, thus avoiding the administration of the first adjuvant to prevent allergic reactions. The advantages of nanoparticles include reducing the injection time, do not have a special injection equipment, do not worry about the exudation of the plasticizer in polyvinyl chloride injection equipment, and this technology can take advantage of the endogenous albumin pathway, so that a larger dose anticancer drugs selectively delivered to the tumor site.Albumin can be combined with albumin receptors of the tumor cell surface glycoprotein 60 (glycoprotein 60, gp60).The activation of gp60 combined with intracellular protein (caveolin) formed cell transfer vesicles -membrane quality membrane capsule through the cell membrane retraction mechanism. The formation of the albumin by the cellular transport mechanism through the endothelium into the extravascular space.Many tumor cells can express high acidic cysteine-rich secretory protein (secreted protein acidic rich in cysteine, SPARC), it’s expression also significantly increased in the malignant transformation process. SPARC in which the tumor over-expression associated with poor prognosis.Studies had shown that the overexpression SPARC in 45-65% of breast cancer in different stages of development. Albumin can specifically bind to the surface of breast cancer tumor cells SPARC [5],albumin nanoparticles to the tumor-specific delivery is achieved through transshipment role of receptor-mediated endocytosis, as well as the specific surface of tumor cells SPARC. Experimental data have shown that paclitaxel albumin nanoparticles compared with the unmodified paclitaxel, offers several improvements including lower toxicity, simplify operations, improve efficacy,no medication before the special treatment, and now has begun to single-agent and multi-drug first-line treatment evaluation with non-small cell lung cancer(NSCLC),ovarian cancer, malignant melanoma aneurysm. Therefore, the development of docetaxel albumin nanoparticles has also become a hot spot of tumor targeting agents.According to the domestic and international related reports, with the nab-paclitaxel technology more development, it has been getting more standout antitumor efficacy and decreasing toxicity. Docetaxel belongs to taxanes chemotherapetic agent.Compared with paclitaxel,it reveals 2 fold better affnity for tubulin, 3 fold higher drug concentration and durg retention in tumor cells, even has a certain active effect for paclitaxel-resistance cells. But the technology of nab-docetaxel is still in the lab.Our laboratory had continued to research and development of docetaxel albumin nanoparticle, the nanoparticle albumin bound technology had obtained national patent.The technological innovation of docetaxel-loaded PEG-albumin nanoparticles has not been evaluated so far.We prepared docetaxel-loaded PEG-albumin nanoparticles (PEG-DANPs) using PEGlyted human serum albumin (HSA) with emulsion-evaporation cross-link method.The best Drug Entrapment efficiency and Drug content were obtained by optimizing synthetic process. The nanopartical size is well distributed with TEM and well stable dispersion is observed via dynamic light scattering method. The in vitro drug release kinetics showed a slower and continuous release pharmacokinetics characteristic. This phenomenon indicated that the PEG-DANPs can maintain high drug concerntration in the plasma, which indicate possible better multi-targeting effect. Compared with docetaxel and nab-docetaxel, PEG-DANPs showed a higher inhibition of the tumor cell poliferation and better inducement of cell apoptosis in vitro. Motility and migration ability assessment with Wound healing assay and Cell invasion assay showed outstanding inhibition of invasion and metastasis in 4T1 cells with PEG-DANPs. The blockage of long distance metastasis molecular mechanism by PEG-DANPs might be due to the downregulation of VEGF, MMP-9, C-met mRNA level. In our study, the therapeutic efficacy using bioluminescence imaging (BLI), a sensitive and non-invasive method successfully used to monitor tumor progression and the metastasis in living animals of breast cancer model with. 4T1-luc cells. The significant effectiveness of inhibiting tumor progress and metatasis has been shown in breast model by PEG-DANPs,according with in vitro. In conclusion, optimizing PEG-DANPs could enhance antitumor effect with active and passive targeting mechanism in vivo.Main methods and result of this research:1、Preparation of PEG-DANPs by emulsion-evaporation cross-link methodPEG-DANPs were prepared using emulsion-evaporation cross-link method.PEG-DANPs could be easily reconstituted in normal saline by mild shaking before application. The colloids solution was light blue in color with opalescence.2、Transmission electron microscopy(TEM) observationThe DANPs and PEG-DANPs morphological observation was examined by TEM. The particles showed spherical shapes with particle size about 160 nm.3、Particle size and zeta potential measurement with dynamic light scattering methodThe PEG-DANPs particle size and zeta potential is 158. 1 ±0.2nm and-17.4 ± 0.6 mV respectively.4、Drug Entrapment efficiency and Drug content with high-performance liquid chromatography (HPLC) method and high-speed centrifuge methodThe drug content in the supernatant after centrifugation was measured by high-performance liquid chromatography (HPLC) method. Drug Entrapment efficiency and Drug content of DANPs was 92.9±0.7%,8.4±0.6%.5、In vitro drug release studyCompared free DT,PEG-DANPs showed a slower and continuous release. This phenomenon indicated that the PEG-DANPs can maintain high drug concerntration in the plasma, which indicate possible better cytotoxic effect.6、Cell viability (MTT assay)MTT assav for the determination of tumor cell half inhibitory concentration of DT,DANPs and PEG-DANPs group is 7.54±0.17μg/mL,4.1089±0.05μg/mL, 3.0089±0.17μg/mL respectively. Both DANPs and DPANPs showed higher or at least the similar cytotoxicity against murine 4T1 breast cancer cell with free DT at equivalent doses. (P<0.01) It was shown that both DANPs and DPANPs has similar dose-and time-dependent cytotoxicity against 4T1 cells. Hence there was significant difference between DANPs and DPANPs.(p<0.05)7、Morphological observation by HE stained mothedThe stained cell were observed and photographs were taken under the light microscope. Compare with Control, free DT or DANPs groups, there was more pyknosis, karyokinesis and a larger cell-free zone in the cells treated with the equivalent PEG-DANPs.8、Apoptosis observation with fluorescence microscopyThe stained cells were observed and photographs were taken under the fluorescence microscopy. PEG-DANPs group can be observed more late apoptosis of the cells.9、Apoptosis determinations and cell cycle analysisThree group of medication groups induce the remarkable cell compared to the control group(p<0.01). The PEG-DANPs group may induce more later periods cell apoptosis compared to other control groups(p<0.01).Cell cycle analysis was assessed by flow cytometry. Treatment group than the control group was significantly inhibited cell proliferation(p<0.01). PEG-DANPs groups can be observed a high proportion G2/M cell cycle block(p<0.05) compare with the DT and DANPs group.10、Motility and migration ability with Wound healing assay and Cellinvasion assayTreatment groups were significantly delayed the wound healing compared with control groups(p<0.01). PEG-DANPs group with more than the other two treatment groups significantly inhibit cell migration(p<0.01).Treatment group has significant inhibition of cell-penetrating ability compare with control group(p<0.01). PEG-DANPs group with more than the other two treatment groups (p<0.01).11、The VEGF, MMP-9 and C-met mRNA expression of 4T1 breast cancer cells by RNA extraction and RT-PCR assayTreatment group has significant low level expression compare with the control group. The expression of PEG-DANPs group significantly lower than DT and DANPs group.12、Preparation and evaluation of 4T1-luc metastatic breast cancer modelMurine breast cancer 4T1-luc cells 1.0×106,1.0×105,1.0×104were inoculated at the right inguinal mammary fat pad of syngeneic Balb/c mice and divided into control, high, medium and low inoculum concentration group. The 4T1-luc2 cells can be detected fluorescence signal on the 2nd day in three spontaneous metastasis in breast cancer models. The cell concentration and enhanced fluorescence signal is positively related. the cell concentration, the higher, longer, The fluorescent signal strength value is higher with the higher cell concentration and the longer time. Three groups of the tumor at different times: the high concentration group d9, the medium concentration of group d11, the low concentration group d16, can be observed to the visible tumor.Adjusting the cell concentration of 1.0×105, divided into normal group, control group and paclitaxel group. The fluorescence signal of the paclitaxel group than the control group significantly decreased, consistent with the tumor growth.13、Bioluminescence imaging (BLI) observationDivided into control group, DT, group, DANPs group, and the PEG-DANPs group,a time-dependent enhancement of bioluminescence average intensity (quanta/sec/mm)was detected with the tumor growth and metastasis in each group mice. In the 32nd day, the bioluminescence intensity of three treatment group significantly reduce compared to the control group. The PEG-DANPs and DANPs group showed a significant reduction in bioluminescence intensity and a significant anti-metastatic effect compare with the control and DT group(p<0.01). The several visceral metastasis occured in DT group , including lung, liver, heart, and bone, and the result verified by Histochemical staining method. PEG-DANPs group showed a significant anti-transfer capabilities, there is no metastasis of vital organs, the bioluminescence intensity is also significantly reduced with DANPs group(p<0.05).14、Examination of in vivo spontaneous lung metastasis and death timeThree treatment groups than the control group significantly reduced the lung metastasis (p<0.01) and PEG-DANP group showed obvious resistance to lung metastasis compared with the other groups (vs DANPs group, p<0.05, vs control and DT group ,p<0.01). Control group metastatic pulmonary nodules are almost full.PEG-DANPs group does not have lung cancer metastasis, DT and DANPs group has a different number of lung metastasis.Control group mice all died at 37.17± 1.25 days and the PEG-DANPs mice all died in 56.50±2.03 days. The survival time could be longer than the other groups by the treatment of PEG-DANPs (vs Control and DT group, p<0.01, vs DANPsgroup,p<0.05).Conclusions of this research:1、Docetaxal loaded PEGylated albumin bound nanoparticles (PEG-DANPs) was prepared based on HSA via emulsion-evaporation cross-link method. The in vitro release studies showed a sustained and continuous release pattern of PEG-DANPs.2、In vitro, PEG-DANPs showed obvious cytotoxic and facilitated more late apoptosis of 4T1 cells compared with DANPs and free DT.3、4T1 cell line survival rate and mobility was strongly inhibited and the expression of VEGF、MMP-9 and C-met mRNA decreased obviously following culture of 4T1 cells with PEG-DANPs.4、The metastatic breast cancer 4T1 model was successfully constructed. In our study,the therapeutic efficacy of PEG-DANPs, DANPs and free DT was compared using bioluminescence imaging (BLI), a sensitive and non-invasive method successfully used to monitor tumor progression in living animals. The advantages of this technique include the visualisation and quantification of disseminated breast tumors without the need of animal sacrifice at each time point of analysis5.It can be demonstrated PEG-DANPs can more effectively inhibit tumor growth,less lung metastasis and longer suvival time in 4T1 breast cancer model. |
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