| Incidence of lung cancer has been increased gradually worldwide in latest 50 years. About 80% of all the patients missed opportunity of surgery when diagnosed. Generally administered clinically, chemotherapy can be used to advanced lung cancer alone or combined with operation and/or radiotherapy.Paclitaxel is a newly discovered broad-spectrum anticancer medicine which is generally applied in recent years. Being a microtubule stabilizer, paclitaxel disturbs the dynamic balance between microtubule protein and microtubule protein polymer, promotes microtubule assembly, inhibits their disassembly and eventually arrests cells in the mitosis.Although paclitaxel has been widely used in the clinic, there are still two obstacles: one is limited resources; and the other is that paclitaxel is a drug with very low aqueous solubility.Nowadays, paclitaxel is dissolved in the mixture of cremophor and absolute alcohol (1:1) before further attenuated. Cremophor itself has been reported to cause serious allergic responses in both animals and human being. In addition, Cremophor can dissolve the plasticizer of transfuse assemblage and eventually cause extra allergic responses. Even after steroids pre-medication to reduce the risk of solvent-mediated hypersensitivity reactions, the incident of allergic response is still as high as 10-30%. So it is necessary to change formulation of paclitaxel in order to improve safety and efficacy.Previous findings showed that pharmacokinetic characters of paclitaxel nanometer particle were quite different from that of cremophor based paclitaxel. With different tissue distribution feature, the former tends to target to tumor. Meanwhile, same treatment efficacy could be achieved by a dose less than equivalent dose with reduced side effects.In 1906, Enrililich first introduce concept of targeting therapy that is drug selectively distributed to areas of disease to achieve higher local concentration, diminish toxicity to normal tissue and enhance efficacy. Tumor cells are different from normal cells from structure to function. Tumor tissue has its unique physiologic nature—EPR (enhanced penetrate and retention) effect. Physiological features of tumor like rapid growth of tumor vessel, shortage of adventitial cell, deformed basal membrane and defect of lymph vessels backstreaming system which enabled rapid-growing tumor tissue get more oxygen and nutrition, increase permeability of tumor vessel and result in EPR effect. Based on rationale above, nanoparticle conjugated anticancer drug that can concentrate to tumor selectively by EPR effect is promising to improve efficacy and diminish toxicity.Based on theory discussed above, amphipathic biodegradation material polylactic acid-macrogol(MPEG-PLA) was chose to prepare conjugate paclitaxel nanometer particle. The reasons are as follows: (1) Since amphipathic feature is preserved after conjugated with paclitaxel, sphere nanoparticle enveloped paclitaxel auto-assemble, which avoid sudden release of paclitaxel. (2) Located in the outer layer of nanoparticle, hydrophilic PEG section enable nanoparticle soluble in both injection water and blood. On the other hand, PEG section could avoid to be removed by immunologic system because of its low immunogenicity and low affinity with protein. Therefore, PEG conjugated nanoparticle could retain in blood circulation for a long time, concentrate at cancer area by EPR effect and improve targeting feature of drug. (3) Being intermediate metabolite of TCA cycle, drug carrier PEG-PLA is the main resource of body energy and essential component, and its absorption and metabolism mechanism is well understood. By the time of drug releasing, PLA is degraded completely and PEG is excreted by kidney, and both of them are proved safe and harmless to human body.PEG-PLA-paclitaxel nanometer particles of different dimensional were prepared; their anticancer effects and paclitaxel release in vitro were studied; also, their distribution and anticancer effects in vivo were investigated in order to explore a new way of target therapy to lung cancer.The main results of the study are as follows:1. It is proved that PEG-PLA-Paclitaxel nanometer particle was successful synthesized by determining the critical micellae concentration of PEG-PLA-OH and PEG-PLA-Paclitaxel by spectrofluorometric method. Observed by scanning electron microscope, morphology of conjugated nanoparticle was proved to be sphere-shaped.2. Studied by dynamic light scattering, distribution of PEG-PLA-Paclitaxel in solution was unity and symmetry and their mean diameter was identical to their molecular mass.3. Studied by HPLC, release rate of paclitaxel from PEG-PLA-Paclitaxel in earlier stage release period (first 3 days) was 30%; release was persistent in the following days, and accumulated release achieved 80%(at day 22).4. There was no significant difference between PEG-PLA-Paclitaxel and Paclitaxel on inhibition effect to proliferation of Lewis lung cancer cell line within 96 hours studied by MTT in vitro. Inhibition ratio of both increased with time and concentration, that is, inhibition ratio is time and concentration depended.5. Observed by inverted microscope, there was no significant difference between Lewis lung cancer cells treated with PEG-PLA-Paclitaxel or those treated with Paclitaxel. Observed by fluorescence microscope after Hoechst 33258 dyeing, cytogenic apoptosis was proved to be induced by either paclitaxel or PEG-PLA-Paclitaxel6.Considering tumor weight and tumor mass, in vivo study proved both PEG-PLA-Paclitaxel and paclitaxel were effective to Lewis lung cancer in mice. It was noted that with the elongation of treatment, PEG-PLA-Paclitaxel were more effective than Paclitaxel.7.Apoptotic cells with apoptosis body in nuclei were found in tissue section of tumor after treated with PEG-PLA-Paclitaxel or Paclitaxel.8.Survival rate of PEG-PLA-Paclitaxel treated mice was higher than that treated with Paclitaxel; and the latter was higher than negative control.9.Observed by laser confocal microscope, 50nm FITC conjugated PEG-PLA particle mainly distributed in liver. To the contrast, 140nm ones mainly distributed in tumor. The major conclusions are as follows:1 . Amphipathic biodegradable PEG-PLA conjugate paclitaxel by covalent bond, and PEG-PLA-Paclitaxel nanoparticle form"shell-core"structure.2.PEG-PLA-Paclitaxel nanoparticle is aqueous soluble sphere-shaped particle.3.Outbreak release of paclitaxel from PEG-PLA-paclitaxel is followed by persistent release.4.There is no significant difference between PEG-PLA-Paclitaxel and Paclitaxel on inhibition effect to proliferation of Lewis lung cancer cell line within 96hs in vitro. Inhibition ratio of both is time and concentration depended.5.Anti-tumor effect of PEG-PLA-Paclitaxel and paclitaxel is related, at least partly, to inducing tumor cell apoptosis.6.With elongation of time, compared with paclitaxel, treatment effect of PEG-PLA-Paclitaxel nanoparticle is superior considering tumor mass, weight of tumor and life span of tumor-bearing mice.7.PEG-PLA whose diameter is 140nm targets to tumor in vivo.The new ideas from the present study are:1. For the first time, biodegradable PEG-PLA conjugated with paclitaxel by covalent bond and formed"shell-core"structure aqueous soluble nanoparticle;2. For the first time, paclitaxel release from PEG-PLA-Paclitaxel nanoparticle was studied; 3. For the first time, inhibition effect of PEG-PLA-Paclitaxel nanoparticle to proliferation of Lewis lung cancer cell line was investigated;4. Mechanism of anticancer effect of PEG-PLA-Paclitaxel nanoparticle was explored for the first time,;5. In vivo distribution of PEG-PLA of different diameter was studied for the first time;6. Treatment effect of PEG-PLA-Paclitaxel nanoparticle to tumor-bearing mice was studied and it was compared with that of palitaxel. |
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