Construction Of CO₂-responsive Micro-nano Drug Delivery System And Its Application In The Treatment Of Asthma

Asthma is a complex respiratory disease characterized by airway inflammation,excessive mucus secretion,and acute hyperreaction,accompanied by increased airway reponsiveness and airway spasm,which ultimately leads to obstruction of alveolar gas exhalation.Inhaled corticosteroids are currently the first choice for clinical treatment of asthma.They can effectively combat allergic inflammation,reduce airway hyperresponsiveness,and improve lung function.However,most of the inhalation products currently on the market are short-acting dosage forms that require frequent and long-term administration,which leads to serious systemic side effects.Therefore,how to control the release of the drug and prolong the residence time of the drug in the lungs is an urgent problem to be solved in the treatment of asthma by inhaled preparations.The particle size of the inhaled preparation determines the location of its deposition in the lung tissue,and CO₂enrichment is the main physiological and pathological feature of the lung tissue.Based on this,this project designed and prepared a CO₂-responsive pulmonary drug delivery micro-nano system,the drug delivery system is modified by an amidine group polylactic acid polylysine（PLA-m PLL）nano core,and yeast glucan microcapsules（YGM）encapsulated on the outside.The large porous sac-like structure of YGM can carry PLA-m PLL/BUD and effectively deposit deep in the lungs.Under the action of alveolar CO₂,PLA-m PLL expands and degrades,releasing the internal drug budesonide（BUD）.In addition,after part of the drug delivery system is taken up by macrophages,the macrophages can be used as a drug library to slowly release BUD to improve the bioavailability of the drug and the therapeutic effect of asthma.The research content of this paper mainly includes the following aspects:1.Construction and in vitro characterization of YGM/PLA-m PLL/BUD.Firstly,PLA-NH₂was obtained by amination of carboxyl polylactic acid initiates ring-opening polymerization of Nε-benzyloxycarbonyl-L-lysine anhydride to obtain PLA-PLL,and then amidinolated to obtain PLA-m PLL,and nanoparticles were prepared by solvent exchange method.The structure and morphology of PLA-m PLL were characterized by hydrogen nuclear magnetic resonance spectroscopy,fourier transform infrared spectroscopy,dynamic light scattering,transmission electron microscopy.The experimental results showed that the potential of PLA-m PLL did not change significantly after the action of CO₂,and the particle size increased by 35 nm.The results of in vitro drug release showed that compared with the control group without CO₂,the cumulative release of BUD in the nanoparticles PLA-m PLL/BUD for 48 h increased from（38.26±5.22）%to（86.37±2.58）%.The release amount of PLA-m PLL/BUD in the YGM/PLA-m PLL/BUD decreased from（58.73±2.86）%to（39.54±2.22）%,and the release amount of BUD increased from（30.59±2.70）%to（80.95±1.29）%.The experimental results show that the YGM/PLA-m PLL/BUD drug delivery system is CO₂-responsive and can achieve sustained release of the drug.2.Research on the role of macrophages.The inflammation of the lungs that accompanies asthma can lead to the accumulation of macrophages.Studies have shown that macrophages can swallow the nano drug delivery system and act as a reservoir for drugs.Therefore,this study investigated the role of micro-nano drug delivery system YGM/PLA-m PLL/BUD and macrophages.Cell uptake experiments showed that with the extension of the action time,the phagocytosis of YGM/PLA-m PLL-RBITC by macrophages continued to increase.The release of drugs from macrophages was then investigated.The results showed that with time,the release of BUD from RAW264.7 cells continued to increase,and the percentage of release at 48 h was（52.57±2.47）%.Experimental results show that the YGM/PLA-m PLL/BUD drug delivery system can slowly release the drug after being taken up by macrophages,which increases the sustained release effect and bioavailability of the drug.3.Study on the distribution and pharmacokinetics of YGM/PLA-m PLL/BUD in vivo.Using small animal live imaging experiments to investigate the distribution of the micro-nano drug delivery system in various organs and tissues.The results showed that after tracheal instillation,the live imaging of mice showed that the fluorescence was mainly distributed in the lung tissue,and YGM/PLA-m PLL could achieve the sustained release of inhaled drugs in the lungs.Then the SD rat was used as the animal model,and the blood concentration of BUD in rat plasma was determined by high performance liquid chromatography.The experimental results showed that compared with the free drug BUD group,the peak time of the YGM/PLA-m PLL/BUD drug delivery system group was significantly increased,and the peak concentration and the area under the curve were reduced.Tmax is extended from（0.25±0.12）h to（4.50±0.57）h,Cmax is reduced from（0.83±0.13）μg/m L to（0.11±0.01）μg/m L,AUC is reduced from（4.09±0.75）μg/m L·h to（2.06±0.75）μg/m L·h.The results show that the YGM/PLA-m PLL/BUD drug delivery system can achieve the local slow-release effect of BUD in the lung tissue,increase the time of action of BUD,and reduce the systemic distribution of the drug.4.The in vivo asthma therapeutic activity of YGM/PLA-m PLL/BUD.OVA was sensitized and challenged to establish an asthma model in female BALB/c mice.Mouse body weight change curve,blood routine,liver function and organ index experiments showe that the drug delivery system has good biocompatibility and no obvious toxicity in mice.The pharmacodynamics of airway resistance,bronchoalveolar lavage fluid cell count and classification count,inflammatory factors,lung tissue sections were investigated.Compared with non-administered asthmatic mice,the airway resistance value of YGM/PLA-m PLL/BUD drug delivery system decreased from（6.93±0.60）cm H₂O/m L/S to（3.31±0.58）cm H₂O/m L/S.The total number of inflammatory cells in bronchoalveolar lavage fluid decreased from（95.17±3.82）×10⁴to（14.25±3.93）×10⁴.This indicates that pulmonary administration of YGM/PLA-m PLL/BUD has a good inhibitory effect on airway hyperresponsiveness and inflammation in asthmatic mice.