| Idiopathic Pulmonary Fibrosis(IPF)is a progressive disease with unclear pathogenesis and lack of effective treatment.Studies have shown that transforming growth factor-β(TGF-β)plays an important role in the progression of IPF,and there is a positive feedback regulation between TGF-β and lactate secretion.In this project,the positive feedback mechanism of TGF-β/ lactate was used as the breakthrough point to design the combined delivery strategy of pirfenidone-cotton phenol,and albumin and mPEG-PLL were used as the main carrier materials,and coaxial electrostatic spray technology was used to prepare the core-shell structure of drug co-loading inhalable microspheres,which could avoid the phagocytosis of pulmonary macrophages.To achieve effective deposition of carrier in the lung and pH-sensitive uptake in pulmonary fibrosis lesions,and play a synergistic therapeutic effect by regulating the TGF-β/lactate pathway and macrophage polarization.The specific content is divided into the following five parts.Part one: ReviewsThis part reviews the research progress of idiopathic pulmonary fibrosis and the mechanism of action of related drugs,and focuses on the introduction of electrostatic spray technology,indicating the advantages of electrostatic spray technology to prepare powder aerosol microspheres.Through the introduction of APIs,it is shown that there are adverse reactions in the traditional administration mode.Through the description of the carrier material,it is shown that the carrier can be used in electrostatic spray,and has less irritation to the body.Part two Synthesis of pH-sensitive polymersIn this part,aldehyde polyethylene glycol monomethyl ether(mPEG-CHO)was synthesized by esterification reaction with polyethylene glycol monomethyl ether and p-formaldehyde benzoic acid as raw materials.The pH-sensitive polymer mPEG-PLL was prepared by amino-aldehyde condensation reaction.The structure and molecular weight of the product were characterized by Fourier transform infrared spectroscopy,hydrogen nuclear magnetic resonance spectroscopy and gel chromatography.The results show that the synthesis of mPEG-CHO and mPEG-PLL is successful.Part three preparation of the powderIn this part,the core-shell structure drug-loaded microspheres with albumin and mPEG-PLL as carriers were prepared by electrostatic spray technology.Firstly,the influence of the type and dosage of auxiliary materials on the morphology of microspheres was investigated by single factor experiment.Secondly,the effect of process parameters on the yield of microspheres was investigated by orthogonal experiment,and the formulation process was optimized.Finally,the physicochemical properties of drug-loaded microspheres were characterized.The drug-loaded microspheres have regular morphology,uniform size and good powder properties.In vitro release results showed that the drug release from the microspheres was slightly delayed,indicating that the hydrophilic carrier did not affect the drug release.Under the condition of pH6.8 and pH7.4,the potential of microspheres was 2.72±0.56 m V and-6.90±1.49 m V,and the particle size of microspheres was 1259.47±99.36 nm and1591.09±56.55 nm,respectively,indicating that the particle size and potential of microspheres could change in weakly acidic conditions.It is pH sensitive.Part four In vitro cell experiment of drug-loaded microsphere powderIn this part,the biocompatibility of blank vector and the inhibitory effect of drugs and microspheres on 3T6 cells were investigated by MTT experiment.The blank vector has good biocompatibility.Pirfenidone and gossypol showed an inhibitory effect on the growth of 3T6 cells,and the combined administration of pirfenidone and gossypol showed a synergistic effect.The microsphere preparation could further improve the inhibitory effect of co-loaded drugs on fibroblasts.Then,the uptake of microspheres in macrophages and 3T6 cells was investigated,and the results showed that PEG modification could reduce the phagocytosis of macrophages.Under weak acid conditions,the uptake of microspheres in 3T6 cells was increased,presumably because the ph-sensitive bond of mPEG-PLL was broken under weak acid conditions,PEG was removed,the potential of microspheres was reversed,and cellular uptake was increased.The mechanism of action of the drug was preliminatively discussed at the cellular level,and gossypol could effectively reduce the lactate secretion of 3T6 cells induced by TGF-β.Part five: Efficacy study of drug-loaded microspheres powder by lung inhalationIn this part,bleomycin was used to establish a rat model of pulmonary fibrosis,and the in vivo efficacy of pirfenidone,gossopol,combined administration group and co-loaded microspheres were investigated.HE and Masson staining experiments showed that compared with single drug treatment,the combined administration group could reduce the collagen content of IPF tissue more effectively,showing a synergistic effect,while the co-loaded microspheres group could further improve the treatment effect and improve IPF.The content of hydroxyproline and lactate was significantly increased in the IPF model group,and the secretion of hydroxyproline and lactate was down-regulated in all treatment groups,and the order of effect was co-loaded microspheres group > Combined administration group > Monotherapy group.The results of immunofluorescence showed that the levels of TGF-β,α-SMA,Collagen-Ⅰ,LDHA and CD206 in lung tissue of rats in the model group were significantly increased.All treatment groups could effectively reduce the levels of α-SMA,Collagen-Ⅰ,LDHA and CD206.Except for the gossypol treatment group,all the other treatment groups showed a down-regulation effect on TGF-β,indicating that gossypol mainly played a role in the treatment of IPF by regulating LDHA.The combined administration group showed a synergistic effect of regulating TGF-β/LDHA,and co-loaded microspheres could further enhance the synergistic effect.In addition,co-loaded microspheres showed the effect of reversing the polarization of macrophages to M2 type.Safety evaluation showed that AST and AUT levels were significantly increased after oral administration of pirfenidone and gossopol,while AST and AUT levels were not significantly changed in the inhaled microspheres group compared with the control group,indicating that inhaled microspheres could effectively reduce liver side effects.The results of HE section and body weight of each tissue also showed that the administration group with inhaled microspheres had better safety. |
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