Polycation-Mediated Pulmonary SiRNA Delivery Toward The Anti-Inflammatory Treatment Of Acute Lung Injury(ALI)

Acute lung injury(ALI)refers to an inflammatory lung disease with damaged capillary endothelial cells and alveolar epithelial cells,and its later stage will become acute respiratory distress syndrome(ARDS)with extremely high lethality.Many inflammatory cytokines play important roles in the ALI process,such as macrophage migration inhibitory factor(MIF),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and interleukin-1β(IL-1β).Compared with the expensive and poor universal monoclonal antibody therapy,gene therapy using small interfering RNA(siRNA)has the advantages of high specificity,high efficiency,and relatively low cost so that to have a good prospect.Cationic polymer is one of the most common gene delivery vectors.It forms stable and positively charged nanocomplexes(NCs)with siRNA through electrostatic adsorption,which can be taken into the target cells.siRNA efficiently silences the target gene in the cytoplasm and destroys inflammatory cytokines expression to achieve the ultimate therapeutic effect.In addition,the suitable way of administration also greatly affects the efficiency of gene silencing.For the ALI model,the intratracheal administration offers higher therapeutic efficacy of siRNA and minimal systemic side effect.However,in the way of intratracheal administration,the presence of mucus layer sets another challenge for siRNA delivery.NCs with large size are quickly removed by mucus.Moreover,positively charged NCs are easily captured in the mucus layer since the highly glycosylated mucin is negatively charged.Thus,the gene delivery efficiency mediated by positively charged NCs is significantly reduced.Therefore,the siRNA delivery system must have the ability to penetrate the mucus layer quickly while ensuring high transfection efficiency at the same time.Herein,we designed two stimuli-responsive gene vectors to overcome the mucus layer barrier,ensure efficient cell transfection,and achieve anti-inflammatory treatment of ALI.In chapter 1,we summarized the current status and related work of ALI,traditional methods for ALI and gene therapy,biological barriers for pulmonary gene delivery,and stimuli-responsive gene vectors.In chapter 2,we designed and synthesized fluorinated,diselenide-cross-linked polyethyleneimine(DSe-PEI-F),which was used to deliver MIF siRNA(siMIF),inhibiting the migration of macrophages at the site of the lesion,and thus inhibiting the inflammatory cascade to achieve anti-inflammatory therapeutic effect for ALI.DSe-PEI-F could form stable NCs with siMIF(～250 nm),which could be efficiently taken into the target cells after quickly penetrating the mucus layer.Compared with non-responsive NCs,DSe-PEI-F/siMIF NCs could be degraded under the stimulation of reactive oxygen species(ROS)with high concentration,thereby quickly releasing siMIF,silencing the MIF mRNA.In LPS-induced RAW 264.7 cells,the gene silencing efficiency of DSe-PEI-F/siMIF NCs was about 85%.In the model constructed by Calu-3 cells,the mucus layer permeability of DSe-PEI-F/siMIF NCs was increased by about 10 times compared to the non-fluorinated NCs.In LPS-induced ALI mice,intratracheally administrated DSe-PEI-F/siMIF NCs could also significantly reduce the expression of TNF-α(～85%)and IL-1β(～75%)cytokines.Moreover,the lung wet/dry weight ratio(～4.2 times),partial pressure of oxygen(～103 mmHg),partial pressure of carbon dioxide(～45 mmHg),and pH(pH≈7.3)basically returned to normal levels.In chapter 3,we designed and prepared a pH-responsive,charge reversible vector for co-delivery of TNF-α siRNA(siTNF-α)and the receptor for advanced glycation end products binding peptide(RBP)to disrupt the expression of TNF-α,thereby achieving anti-inflammatory treatment.Dendritic poly-L-lysine(DPLL)was synthesized via ring-opening polymerization(ROP)of N-carboxyanhydride(NCA)as initiated by G3-PAMAM and used to form positively charged DPLL/siTNF-α(DsT)binary NCs.RBP-cis-aconitic amide(RBP-CA,RC)was synthesized via ROP of cis-aconitic anhydride as initiated by RBP,and the negatively charged RC/DPLL/siTNF-α(RCDsT)ternary NCs was obtained by wrapping the RC layer onto the DsT NCs.RCDsT NCs quickly penetrated the negatively charged mucin glycoproteins through electrostatic repulsion,and RC realized charge reversal in the slightly acidic environment of the inflammatory site.Positively charged DsT NCs could be efficiently taken into macrophages,silencing the expression of TNF-α,while RBP bound to RAGE on the inflamed macrophage membrane,thereby achieving combinational anti-inflammatory treatment for ALI.In LPS-induced RAW 264.7 cells,the RCDsT NCs had a superior silencing efficiency of TNF-α cytokine(～70%),achieving combinational anti-inflammatory effect of siTNF-a and RBP.In the model constructed by Calu-3 cells,the mucus layer permeability of RCDsT NCs was increased by about 7 times.In mucin aggregation study,RCDsT NCs effectively reduced the adsorption with mucin by about 60%.In LPS-induced ALI mice,intratracheally administrated RCDsT NCs could also significantly reduce the expression of TNF-α(～85%)and IL-6(～77%)cytokines.Moreover,the lung wet/dry weight ratio(～4.4 times),partial pressure of oxygen(～116 mmHg),partial pressure of carbon dioxide(～48 mmHg),and pH(pH≈7.4)basically returned to normal levels,indicating that RCDsT NCs could restored the pulmonary functions of ventilation.Finally,we summarized this paper and looked forward to the future work in the field of this research.