| BackgroundLung cancer is one of the cancers with highest mortality rate in recent years, andthe prevalence rate of which is in a sustained rising trend. With the increase in lungcancer patients, effective treatment for lung cancer becomes the focus and difficultyfor more and more researchers. Currently, the main strategy of lung cancer treatmentincludes surgery, chemotherapy and radiation therapy.We already know that although radiotherapy has the advantage in local damagetreatment, there are still injury to DNA in normal tissue cells. It is not easy to curecancer thoroughly, and curative effect is not significant. Chemical drug therapyinhibiting tumor cells while making huge systemic side effects, pain to the patient.Some of them cannot be used as a first-line drug, since they are more expensive. Forgene therapy, one of the biological treatment, is often applied as supplement ofchemotherapy and radiation therapy. As an important non-specific anti-tumorantibiotic, Doxorubicine (Dox) is applied to the treatment of ovarian cancer, breastcancer, lung cancer, hepatoma and eukemia. Tumor suppressor gene p53plays a keyrole in the formation process for many cancers, such as head and neck cancer, livercancer, ovarian cancer etc. in II, III clinical trial studies. For the treatment of lungcancer, more researchers are committed to develop safe, efficient, no (low) sideeffects system of pulmonary inhalation in situ. Inhaled porous microspheres is one ofthe hot topics, so far, the main carrier material is biodegradable polymerpoly-(lactic-co-glycolic acid)(PLGA) which is safe and nontoxic.Cancer is the result of an imbalance of cell proliferation and apoptosis, apoptosisis blocked in tumor cells, tumor cells with unlimited proliferation and running mok,is the main cause. In the process of apoptosis, mitochondria is a very importantprocessing section, which plays a vital role in the concentration of intracellularcalcium ion, maintaining mitochondrial membrane potential and cytochrome C releasing.The redox in aerobic organisms keeps balanced, which is an important guaranteefor maintaining the body’s necessary physiological activities. When it is subjected tointernal and external stimuli, the original redox balance has been broken, oxidativestress will be produced by the cells. Ordinarily, reactive oxygen species and otherkinds of free radicals are generated. Due to the presence of the body’s defense systemwith five defensive line for antioxidant and free radical scavenging, cytochromeoxidase, antioxidant enzymes, antioxidant (scavenger), repair of free radical damageand apoptosis. Therefore, the reductive system will protect against oxidative stress inthe body. If the reduction system of the tumor site is destroyed, the oxidative stresswill be remained in order to initiate apoptosis via the mitochondrial pathway.Searching for pulmonary inhalation therapy with PLGA porous microspherescarrier, to achieve a Dox and p53tumor suppressor gene co-delivery system is animportant problem to be solved in this study. Whether the Dox and p53tumorsuppressor gene co-delivery system can be effective in human lung cancer cell lineA549cells plays a key role for pulmonary inhalation therapy. If effect of this systemfor pulmonary inhalation is better, whether the functioning molecular mechanismbeing related to ROS-mediated mitochondrial apoptosis or not is a deeper explorationfor the essentially biological efficacy of pulmonary inhalation with this co-deliverysystem.PurposeDevelopment of Dox and tumor suppressor gene p53porous microspheresco-delivery system (PMS-Dox/p53) for lung cancer inhalation.SignificanceProvide a new method for the treatment with synergistic effect of chemotherapyand gene therapy of lung cancer inhalation.MethodIn this article, PLGA was prepared as carrier material, porous microspheresPMS-Dox/p53was prepared simultaneously carried with Dox and p53-PEInano-particles. The author established the efficacy of Dox with HPLC, analyzed many factors (concentration of porogen, concentration of PLGA, homogenization speed)impacting on the appearance of the structure of porous microspheres, optimized theprescription with a series of characterization such as particle size, zeta potential,encapsulation efficiency and in vitro release. The porous microspheres co-deliverysystem in this article would effect on human pulmonary carcinoma cell line A549, andthen the author examined the effect of morphology in A549cells with PMS-Dox/p53(nucleus morphology, cell migration) and situation of apoptosis. Simultaneously theauthor used Western Blot to detect the impact of PMS-Dox/p53to the expression ofapoptosis-related protein factor in A549cells, such as P53, Caspase3, Caspase8,Caspase9, Bcl-2and Bax, and also detected the activity of Caspase3, Caspase8,Caspase9. Further exploration was made as the PMS-Dox/p53group induced theapoptosis of A549cells, the mitochondrial pathway related factors transformed,including content of ROS and hydroxyl radicals, mitochondrial membrane potential,concentration of intracellular Ca2+, intracellular cytochrome C location and variousantioxidant enzymes activity. We discussed the molecular mechanism of apoptosis inA549cells induced by PMS-Dox/p53.Result1. To determine the best HPLC condition for Dox is Ascentis dC18column, themobile phase was0.01M sodium acetate: methanol=30:70, adjusted the pH to4.0with acetic acid, the detection wavelength was254nm. To determine the optimalprescription condition for non-load microspheres (Blank) was10%concentration ofporogen,25%concentration of PLGA,3000rpm homogeneous speed. Preparation ofround configurated porous microspheres,20μm uniform particle diameter with clearporous structure.2. When the p53-PEI nano-particle was loaded, the mass ratio for PEI and p53plasmid was0.4. In this condition, p53-PEI nano-particle had183.4±90.1nmdiameter and17.4±9.8mV zeta potential.3. At the same time, we successfully loaded the chemotherapy drug Dox andtumor suppressor gene p53into porous microspheres, prepared it into PMS-Dox/p53.The diameter of PMS-Dox/p53was22.87±11.79μm and potential of porous microspheres was slightly positive. The entrapment efficiency of Dox and p53-PEInano-particle was88.2±1.7%and36.5±7.5%. The material of porous microspherescarrier was secure, non-toxic, biodegradable, sustained release for seven days afterdrug-loaded. Among them, the release of Dox was later than p53-PEI nanocompositerelease.4. When the drug-loaded porous microspheres was acting on A549cells,PMS-Dox, PMS-p53, PMS-Dox/p53groups of porous microspheres decreased thesurvival rate of A549cells, and also induced apoptosis. With observation of nuclearmorphology via DAPI staining, cell panorama via crystal violet staining, cellmigration rate via cell scratch test, a series of morphological study found that everygroup of drug-loaded porous microspheres was found chromatin condensation,nuclear shrinkage, edge feather and migration decease.5. Drug-loaded porous microspheres induced A549cells apoptosis, and alsocaused the expression level of a series of apoptosis related factor transformed,including tumor suppressor p53up-regulated, pro-apoptotic factor Bax up-regulated,Caspase3, Caspase9up-regulated, anti apoptosis factor Bcl-2down-regulated. In theactivity detection of Caspase3, Caspase8, Caspase9, the activity of Caspase3,Caspase9increased, the activity of Caspase8was almost the same, which confirmedthat the drug-loaded porous microspheres inducing A549cells apoptosis may berelated to the process of mitochondrial apoptosis pathway.6. Iconic indicators of mitochondrial pathway was detected. We found whendrug-loaded porous microspheres was acting on A549cells, it would cause ROSincreasing rapidly. Inner mitochondrial membrane potential decreased, concentrationof intracellular Ca2+increased. The activity of Antioxidant enzymes SOD, CAT, GPxdecreased, while the antioxidant GSH content decreased. Cytochrome C released frommitochondria into the cytosol. Surface drug-loaded porous microspheres in A549cellsis indeed closely related to the ROS and mitochondrial apoptosis. And the effect ofPMS-Dox/p53group was most obvious, it had great advantages on lung cancerinhalation. We speculated that it is related to a synergistic effect betweenchemotherapy Dox and tumor suppressor gene p53. ConclusionThis paper is about the porous microspheres preparation, drug loading and theeffect on A549cells, launching a creative work and systematic research. The mainconclusion as follows:1. We prepared an uniform structure, round appearance, particle size of20μm,positive potential, efficient standard of entrapment, suitable for pulmonary inhalationporous microspheres co-delivery system PMS-Dox/p53.2. Carrier material is biodegradable polymer PLGA, secure, non-toxic, can bebiodegradable in vivo, consistent with the requirement of pulmonary inhalation, thisporous microspheres can achieve sustained release effect.3. PMS-Dox/p53inhibits the survival rate of A549cells, induces apoptosis ofA549cells. And it influences the factors related to apoptosis in A549cells,pro-apoptotic factor is up-regulated and anti-apoptotic factor is down-regulated,compared with other groups, it has the most obvious effect on apoptosis.4. We determine that porous microspheres co-delivery system PMS-Dox/p53inducing apoptosis is mediated by ROS mitochondrial pathway.To sum up, this study of PMS-Dox/p53prepared can provide a new method totreat lung cancer with the chemical therapy and gene therapy of inhaled drug synergy. |
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