An Inhaled Oridonin-loaded Porous Microparticle For The Local Treatment Of Primary Non-small Cell Lung Cancer

Lung cancer is a tumor with high mortality. NSCLC account for about 80-85% of all of lung cancers. Traditional chemotherapy usually leads to serious side effects due to the systemic distribution of drugs. Pulmonary drug delivery is a non-invasive method for the location treatment of lung cancer. It can decrease dosage and body toxicity.Oridonin is used as the anticancer agents. However, its clinical application is highly limited due to its poor water solubility and low bioavailability. This study showed two preparation technologies of large porous microparticles loading oridonin for the directly local treatment of primary NSCLC through airway. The key problem could be addressed only with a novel strategy where a large porous microparticle(LPMP)keeps a relatively apparent large diameter but with low density and small aerodynamic diameters. The formulation and preparation method of the microparticles were screened. The characteristics and drug release of the microparticles were investigated. Finally, the high anticancer effect of the microparticles was demonstrated on the primary NSCLC rat models and the mechanism was explored.The followings are the research details:1. Preparation of emulsion solvent evaporation method for oridonin-loaded large porous microparticles(LPMPs)The formulation and preparation method of oridonin-loaded LPMPs were screened.The microparticles occupy the large porous structure and low tapping density,resulting in appropriate aerodynamic diameters, high lung deposition, and escaping from phagocytosis due to the large sizes, i.e. ideal lung-inhaled particles. The optimal formulation of oridonin-loaded LPMPs was prepared from 1.5% ammonium bicarbonate solution, 35% PLGA solution, and 1% PVA solution, where ammonium bicarbonate and PVA were eliminated from the final microparticles that containing9.3±0.1% oridonin with a high encapsulation efficiency of 81.5±1.0%. The oridoninloaded PLGA LPMPs were the smooth spheres with many small pores on the surfaces according to the scanning electron microscopic(SEM) image. Most of the LPMPs had the microscale diameters about 10 μm. Furthermore, after artificially grinding of LPMPs, a lot of cavities exhibited in the inner spaces. The geometric diameter of LPMPs was 11.6±2.3 μm(D50) according to the measurement of laser light scattering method. Therefore, the LPMPs showed a very low mean tapped density of0.057±0.014 g/ml and a small mean aerodynamic diameter of 2.7±0.3 μm. The smooth spherical surface of LPMPs led them easily flowable with a small mean repose angle of 26.5±4.1°. It met the demand of lung delivery.2. Preparation of electrospinning method for oridonin-loaded electrospun porous microparticles(EPMs)The formulation and preparation method of oridonin-loaded LPMPs were screened.The EPMs occupy the porous structure and low tapping density, resulting in appropriate aerodynamic diameters. The EPMs formulation was screened by good modality of porous structure. The results was that the optimal bank formulation of EPM was prepared from 1.5% ammonium bicarbonate solution, 6% or 8%PLGA solution, and oil: water(10:1), where ammonium bicarbonate was eliminated from the final microparticles that containing 5.86±0.2% oridonin with a relatively high encapsulation efficiency of 58.59±2.0%. The geometric diameter of EPM was 5.23μm(D50) according to the measurement of laser light scattering method. Therefore,the EPM showed a very low mean tapped density of 0.15±0.02 g/ml and a small mean aerodynamic diameter of 2.1±0.1 μm. The EPM led them easily flowable with a small mean repose angle of 29.37±6.6°. It also met the demand of lung delivery.3. Determination qualityof oridonin by HPLCHPLC method was established to calculate the quality of oridonin in the formulation.Through the methodology validation of the method is accurate and reliable. The rate of recovery and linearity were acceptable.4. Evaluation of oridonin-loaded LPMPs and EPMs in vitro / in vivoOridonin showed a relatively rapid release from the LPMPs with approximately 80%release. A Ritger-Peppas model can be used to describe the release profile of oridonin from the LPMPs, i.e. the oridonin release mechanism was mainly due to the combination of diffusion and PLGA erosion of the PLGA LPMPs. The LPMPs could not be uptake in 12 h. It suggested that oridonin can release to the surroundings and having many opportunities to enter the cancer cells before alveolar macrophage uptake. The most of the LPMPs deposited in the deep site of lung according to the 3D imaging in vivo. The lung tissue section images further demonstrated the high lung deposition efficacy of LPMPs compared with the conventional microparticles. The result was the same as lung deposition in vitro.Oridonin showed a gradually release from the EPM with approximately 70% release within 120 h. The sustained oridonin release would make it having many opportunities to escape from EPM through the pores to enter the cancer cells and achieve a sustained accumulation in the cells before the eroded microparticles. Thus it could decrease frequency of using oridonin. A Ritger-Peppas model can be used to describe the release profile of oridonin from the EPM, i.e. the oridonin release mechanism was mainly due to the combination of diffusion and PLGA erosion of the PLGA EPM.The 3D imaging in vivo and lung tissue section images demonstrated the high lung deposition efficacy of EPMs.5. Establishment of the primary NSCLC rat modelsThe 3-Methyl cholanthrene(MCA) and diethyl nitrosamine(DEN) were induced to establish the primary NSCLC rat models. The screening of carcinogen dosage was in order to shortenthe time of establishing the primary NSCLC rat models. The progression of primary NSCLC rats was proved by tunel and methylation analysis.The type of NSCLC was known as squamous cell carcinoma through result of pathology section.6. Effect of oridonin-loaded LPMPs and EPMs on the primary NSCLC rat modelsThe oridonin showed anti-NSCLC effect after pulmonary delivery. LPMPs could improve anti-NSCLC effect in oridonin. Improvement of lung cancer cell apoptosis may be the major mechanism. The oridonin PLGA LPMPs is a promising dry powder inhalation for the local treatment of lung cancer.The anti-NSCLC effect of oridonin-loaded PLGA EPMs after pulmonary delivery was evaluated initially. EPMs could also improve anti-NSCLC effect in oridonin. The technology of electrospinning method is a promising preparation of dry powder inhalation for the local treatment of lung disease.