| Lung cancer is more and more common in recent years, almost more than one million patients have been diagnosed with lung cancer each year all over the world. The incidence and mortality of lung cancer is the first place in the city, even in the countryside, it is the second only after the stomach cancer. NSCLC accounts for 75%～ 85% among the total number of lung cancer, and elderly patients whose age more than 65 years accounted for more than 50%. It is always at the late stage when patients were discovered (stage III or IV) and the five years survival rate is only 12% to 15%, so the patients lost the best opportunity of surgical treatment.For elderly advanced NSCLC patients, the chemotherapy is still the first choice of treatment, it is a relatively effective treatment for elderly NSCLC patients who can’t tolerant to surgical treatment. It can effectively reduce the progress of lung cancer and the recurrence, enhance the effect of clinical treatment, prolong patient survival and improve their quality of life.In recently, there are some chemotherapy regimens and drugs for elderly NSCLC patients, such as cisplatin, VP-16, gemcitabine, paclitaxel and docetaxel, etc. However, there is lack of a mature and effective way for NSCLC because most of the elderly patients refuse or unable to complete the treatment. So how to choose a drug with good effect and little side reaction is a hot spot in tumor research. Docetaxel is a kind of cell cycle specificity anti-tumor drugs which apply to cells at M phase. It is a relatively new sort of anti-microtubules taxane drugs, and it is the only one approved first-and second-line chemotherapy for NSCLC drug treatment by the U.S. food and drug administration (FDA) and the EU. According to the existing clinical data of current studies, DTX has been proved very effective for NSCLC.However, the low solubility in water for DTX is a very difficult problem which severely limited its clinical application. Polysorbate 80 (Tween 80) in saline solution was used to enhance the solubility of DTX in commercial products, such as Taxotere (?) and Aisu (?), it can induce hemolysis reaction and allergic reaction which is very common in clinical use. DTX in these two commercial products (Taxotere (?), Aisu (?))showed nonspecific toxicities to normal organs which led to intolerable side effects such as peripheral neuropathy, hemolytic reaction and hypersensitivity reactions. Furthermore, the short elimination half-life of DTX in commercial products cause to more frequent injection in clinical which increase great inconvenience and pain for the patients. Therefore, remove and improve the above-mentioned issues in these commercial products of DTX get more and more attention.Biodegradable albumin nanoparticles have received quite a lot of attention in cancer targeted therapeutics during the past few decades, The previous reports have proved that drug-loaded polymeric nanoparticles could accumulate in certain tumors more efficiently than other carriers by enhancing permeability and retention (EPR) effect, its another advantage is long circulation half-life and significantly lower systemic toxicity which is more superior to conventional drug formulations.The molecular formula of polyethylene glycol (PEG) is H (CH2CH2O) nOH, its molecular structure composed of epoxy ethane polymerization, with the physical and chemical properties of pH neutral, water-soluble polymer, highly hydrophilic, etc. PEG physical and chemical properties are as follows:non-toxic, non-immunogenicity, good biocompatibility, coupling most of the protein polypeptide, small molecule drugs and liposome preparations by covalent or non covalent bonds, change the molecular structure of the drug thereby it could improve the pharmacokinetic and pharmacodynamic properties of drugs. It is one of medicinal synthetic polymer injections which can be used for the body approved by FDA.There are many advantages of PEG drugs such as:(1) Extending biologic half life of drug, enhancing long-acting and sustained-release effect; (2) Improving the solubility and stability of the drug; (3) Reducing immunogenicity and antigenicity; (4) Reducing enzyme degradation; (5) Enhancing the targeting function of drugs; (6) Reducing the toxicity of some drugs, etc. Based on the above advantages, in this experiment we chose PEG to formulate polymeric nanoparticles because of its excellent biocompatibility and biodegradability properties, then we make a comparison between Aisu(?), DANPs and PEG-DANPs to see their effect against NSCLC.In this study, a emulsion-evaporation cross-link method was used to prepare DANPs and PEG-DANPs for intravenous injection. Physicochemical characteristics, in vitro release properties, hymolysis and in vitro cytotoxicity against A549 lung cancer cells of the Aisu(?), DANPs and PEG-DANPs were studied and compared in this paper. The in vivo pharmacokinetics and the in vivo anti-tumor and metastases resistance effects of these three drugs were also assessed in A549 tumor-bearing nude mice. Results obtained in our study demonstrated that PEG-DANPs were a quite promising modality for NSCLC than DANPs and Aisu(?).Methods and Results1. Preparation of PEG-DANPs in emulsified volatile crosslinking method.DTX was dissolved in chloroform and ethanol to form solution A, albumin was dissolved in sterile water to form solution B. The solution A and B was mixed and stirred by homogenate machine 5 min to form raw milk, move the raw milk into high pressure homogenizer, under the 20000 psi for 12 cycles. The chloroform of mixture was eliminated by using rotary evaporator for 25 min and followed by filtration through a 0.22μm filter.DANPs and mPEG (20000 kDa) was added to the solution of boric acid buffer (0.1mol/L, pH9.0) according to the ratio of 3:1 for stirring and the reaction was terminated via adding glycine (lmg/mL) 3h later. Unbound HAS and PEG were removed by ultrafiltration (MWCO:70KDa).Determination via PAGE gel electrophoresis:DANPs and PEG-DANPs were determinated via PAGE gel electrophoresis with iodine staining and mas blue staining. The results showed that PEG-DANPs had been syntheticed successfully.2. Dynamic light scattering method to determine the size and potential DANPs and PEG-DANPsThe mean diameter, size distribution and zeta potential of the DANPs and PEG-DANPs were measured using a Malven Nano ZS laser particle analyzer. The analysis was performed three times and the average values were accepted. The DANPs had an average particle size of 163.4±3.76 nm, a zeta potential of-19.4±0.18 mV; the average particle size of PEG-DANPs is 169.1±2.36 nm, zeta potential is-18.2±0.21 mV.3. Observing the form of DANPs and PEG-DANPs with Hitachi H-7650 electron microscopy (TEM).The morphologies of the DANPs and PEG-DANPs nanoparticles were analyzed by transmission electron microscopy (TEM). The general process is a suspension of DANPs/PEG-DANPs with a concentration of 2 mg/mL was applied dropwise onto a 400-mesh copper grid coated with carbon and negatively stained with 1% w/v phosphotungstic acid solution. The morphology images of TEM indicated that DANPs and PEG-DANPs were spherical with smooth surfaces.4. Determination of DANPs and PEG-DANPs drug loadings and the coating rate via high performance liquid chromatography (HPLC) and high speed centrifugation.The concentration of DTX was assayed on an Agilent 1200LC HPLC system. The mobile phase consisted of a mixture of acetonitrile and water (55:45, v/v) delivered at a flow rate of 0.5 mL/min. The injection volume was 20μL and the wavelength was set at 230 nm. The column temperature was 25℃. The drug loading of DANPs is 8.71± 0.98%, and an encapsulation efficiency of 93.58±0.86%, and PEG-DANPs is 8.72± 1.05% and 95.4±5.5%, respectively.4. In Vitro Drug Release.In vitro release of Aisu(?)、DANPs and PEG-DANPs were assayed using dialysis diffusion method. Typically,0.5 mL of Aisu(?)、DANPs and PEG-DANPs at DTX concentration of 1 mg/mL was sealed into a dialysis bag. The bag was immersed in PBS at 37℃ with a shaking rate of 100 rpm. At predetermined time points,0.2 mL samples were taken from the bags and the same volume of fresh medium was added. Each sample was centrifuged at 10000 rpm and the supernatant was assayed by HPLC with the same condition described before. From the result we could see that PEG-DANPs showed a slower and continuous release of the whole process than DANPs and Aisu(?).5. Hemolysis Test.Tresh whole blood was collected from male guinea pigs. Stir quickly to take out fibrousprotein from the blood, add 0.9% saline and centrifuge at 2000 rpm for 15 min until the supernatant did not turn red. The prepared mixture was made into 2%(v/v) red cell suspensions. Aisu(?), DANPs and PEG-DANPs were diluted to different concentrations with 0.9% saline. Take the same volume of drug solution and the red cell suspension to make a mixture at 37℃ for 1 h. Finally, detect the supernatant at 576 nm. According to result, we see that Aisu(?) displayed much more toxicity toward red blood cells (RBC) than DANPs and PEG-DANPs at the same concentration. The results indicated that PEG-DANPs caused the lowest hemolysis between the three drugs.6. Pharmacokinetics.Twelce male Bal B/c mice were divided into three groups which were injected with Aisu(?), DANPs and PEG-DANPs (20mg/kg) through the tail vein. Collect 0.3 mL blood from the carotid artery into heparinized tubes at predetermined time points. Blood was immediately centrifugated to isolate the plasma. Mix 100μL plasma and 3 mL supernatant together and centrifuge, then take the organic phase to repeat the above process, use HPLC for determination. In our study, after the injection of DANPs and PEG-DANPs, the DTX concentration was still measurable after 24 h, the concentration of PEG-DANPs is higher than DANPs. On the contrary, the Aisu(?) was not detectable even after 12 h. In conclusion, the PEG-DANPs significantly enhanced the half-life of DTX.7. Determination of Cell inhibition via MTT method.The in vitro cytotoxic activity of DANPs and PEG-DANPs was evaluted by the MTT assay. Briefly, the A549 cells (8×104 cells/mL) were seeded in 96-well plates and incubated for 24 h to allow cell attachment. The cells were then treated with a series of PBS, Aisu(?), DANPs and PEG-DANPs, at different concentration. At the determined incubation-time points of 24h,48h and 72h, MTT was added and incubated for 4 h, and then DMSO was added to dissolve the formazan crystals, and the plate was gently shaken for 10 minutes. At last, the optical density (OD) was measured at 490 nm by Synergy H1m Monochromator-Based Multi-Mode Microplate Reader. From the result, we know that PEG-DANPs accelerated cellular uptake of the drug and induced higher cytotoxicity in cancer cells than Aisu(?) and DANPs, especially at lower DTX concentrations (0.001-0.1μg/mL). However, A549 cells were more sensitive to Aisu(?) than DANPs and PEG-DANPs at higher concentrations (1-100μg/mL). In 72 hours, Aisu(?) was hardly checked in the blood so that the inhibition rate of cell is almost impossible to detect.8. Cell apoptosis assay.After 48 h of incubation in the exponential stage, A 549 cells seeded in 12-well plates were treated for a further 48 h with PBS, Aisu(?), DANPs and PEG-DANPs, respectively. After treatment and a series of operations, apoptosis was then analyzed using a FACScan cytometer. PEG-DANPs (50.65%) increased late apoptosis in A 549 cells compared with DANPs and Aisu(?)(25.85% and 45.00%)9. Cellular uptake of coumarin 6(C6)-loaded SPM (C6-SPM).Briefly, the polymers and excess C6 were co-dissolved in CHCl3 and a thin film was formed by the evaporation of CHCl3. A549 cells in exponential-stage growth were incubated with C6-SPM at 37℃ for 5,10,20, and 30 min respectively, then the plate was rinsed three times with cold PBS and fixed with 4% paraformaldehyde. Finally cells were observed by confocal laser scanning microscopy. Images were examined using differential interference contrast and C6-SPM was recorded with the green channel (C6) with excitation at 488 nm. CLSM images at 5 and 10 min showed that C6-SPM fluorescence (green) was closely located around the membrane, indicating that C6-SPM had not been internalized into A 549 cells. However, when the incubation time was extended to 20 min, C6-SPM was successfully internalized into A 549 cells. The figures demonstrated that PEG-DANPs could be absorbed easier into the nucleus than DANPs.10. Tumor Study and In Vivo Antitumor Efficacy.All experimental procedures were performed in conformity with institutional guidelines and protocols for the care and use of laboratory animals. We chose 40 BalB/c mice to divide into two parts, one part will be established the transplantation tumor model to observe the inhibition of drugs for transplantation tumor and weight changes of nude mice; the other part will be established the situ carcinoma model to observe the inhibition of drugs for situ carcinoma and metastasis. Each part contains 20 mice and they were equally divided into four groups, respectively negative control group (glucose injection group), positive control group (Aisu(?) group), DANPs group and PEG-DANPs group with 5 animals of each. The lung caner A549 cells were suspended in BD Matrigel, and the mice in each group were implanted with 3×106 cells in alar or in lung. When the tumor volume in transplantation tumor group reached to about 120 mm3, the mice were treated 4 times at 7-day intervals with 5% glucose injection group (negative control), Aisu(?), DANPs or PEG-DANPs at the same time in both two parts, respectively. All formulations were injected intravenously via the tail vein at a DTX dose of 20 mg/kg. The body weight and tumor volume were measured simultaneously. Tumor volume was calculated using the equation of V=w2×1 /2. Here w and 1 are the width and length of the tumor.48 hours after the last treatment, the mice were sacrificed. The tumors with lung and the other major organs (including heart, liver, spleen, kidney) were removed, fixed in 10% formalin solution, and subjected to paraffin embedding for HE staining.The body weight of the mice in the negative control groups was basically unchanged or slightly increased, while the mice in the drug groups all lost weight in the process of the treatment. However, the reduction range in the Aisu(?) group was more significant than other two groups, suggesting severe systemic toxicity in addition to tumor toxicity. It was found that all the tumor volumes treated with Aisu(?), DANPs and PEG-DANPs were much smaller than those of negative control groups treated with the same dose, and the tumors of PEG-DANPs groups were obviously smaller than those of Aisu(?) groups, indicating that PEG-DANPs is the most effective to inhibit tumor growth among the three.A typical tumor cellular morphology was seen under the microscope in the negative control groups both in transplant model and in situ. No matter from the tissue of tumor or lung, we could see that the tumor cells were of different sizes with a lot of deeply blue stained nuclei, and the nuclear division was significant. There was also a loss of polarity, tightly packed cells with ill-defined nuclei and visible focal degeneration or necrosis of cancer cells. This showed that the model had been established successfully. In the PEG-DANPs groups, the number of cancer cells was significantly reduced, the color of nuclei was stained red rather than light blue in DANPs and Aisu(?) group, and large necrosis area could be seen without any nuclear division.Extensive metastases were found in the livers of control group mice, and there were large tumor nests, we could also see the liver metastases both in Aisu(?) and DANPs treated mice which were significant smaller than control groups. In contrast, liver tissue from PEG-DANPs treated mice showed barely measurable levels of tumor cells but only few neutrophils infiltrated by tumor cells, these results suggest that PEG-DANPs could suppress metastases in other organs such as liver more effectively than DANPs and Aisu(?).11. Observe the survival rate of the nude mice after treatmentSelect another 32 of nude mice to establish the transplantation tumor model and divide them into negative control group (group glucose injection), positive control group (Aisu(?)), DANPs and PEG-DANPs group, the mice were treated until to natural death, observe the survival rate and draw a rate curve. The results showed that the mice which were treated with PEG-DANPs can survive longer than the other groups.Conclusion1. Successfully prepared the docetaxel loaded PEG-albumin nanoparticles;2. The PEG-DANPs had an appropriate particle size, stable distribution, good drug loadings and coating rate. Also, it had an obvious sustained release effect and lower damage effect to the body’s red blood cells;3. The PEG-DANPs can easily enter the A549 cells, it could effectively inhibit the proliferation of A549 cells and induce the apoptosis of the cells;4 The PFG-DANPs can effectively inhibit the growth of NSCLC transplanted tumor and situ carcinoma, it can suppress metastases more effectively and prolong the survival of the mice, furthermore, its toxic effects on the body is small. |
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