| Acute lung injury(ALI)and acute respiratory distress syndrome(ARDS),which is in a critical phase progressing from ALI,can result from various intra-pulmonary or extra-pulmonary causes which are non-cardiac.ALI/ARDS is pathophysiologically an acute inflammatory response syndrome characterized by progressive dyspnea and refractory hypoxemia,which can cause respiratory dysfunction or respiratory failure.We usually divide the causes of ALI/ARDS into two categories,including direct factors and indirect factors,both of which can lead to lung injuries.The direct factors mainly include:various types of pulmonary infection,chest or lung trauma.The indirect factors include:shock,sepsis,systemic inflammatory response syndrome,diffuse intravascular coagulation.Uncontrolled inflammatory responses of the lung and/or uncontrolled systemic inflammatory responses are the most important pathogenesis of ALI/ARDS.The cytokines involved in the inflammatory response of ALI/ARDS include interleukin-1(IL-1),interleukin-6(IL-6),interleukin-8(IL-8),interleukin-12(IL-12)and tumor necrosis factor-a(TNF-a),which are also named pro-inflammatory cytokines(PIC).The cells involved in the inflammatory response of ALI/ARDS include polymorphonuclear leukocytes(PMN),macrophages,alveolar epithelial cells,vascular endothelial cells,etc.RNA interference(RNAi)is a phenomenon of gene silencing,in which the high-efficiency and specific degradation of homologous mRNA was induced by double-stranded RNA(dsRNA)molecules that share homology with the sequence of target genes.Molecules that mediate RNA interference include siRNA,microRNA(miRNA,miR)and shRNA.At present,there are two sets of vector systems for mediating RNA interference,comprised of viral vectors and non-viral vectors.Non-viral vectors,especially nanomaterial carriers,are receiving increasing attention from researchers at home and abroad.As an efficient gene-silencing technology,RNA interference technology is one of the greatest scientific and technological advances in this century.It has been widely used and rapidly developed in the field of basic biomedical research for more than a decade,which provides new means and hope for the treatment of many diseases.Selective silencing of the expression of inflammatory-related cytokines in alveolar macrophages by RNA interference technology can effectively inhibit the inflammatory response and thus achieve the purpose of treating ALI.Therefore,we used TNF-a mRNA in alveolar macrophages as a target of RNA interference for anti-inflammatory treatment of ALI.Most cationic polymer-based nano-carriers that have been developed exhibit some cytotoxicity due to their inability to degrade in vivo,the higher surface potentials of which also result in their quick removal out of the body.Aiming at these problems,this study designed a bioreducible nonlinear nanomaterial based on a cationic polymer,poly(P-amino ester)s(BP-S),to achieve the controlled release of siRNA with reduced cytotoxicity.In addition,we encapsulate the nanocomposite in carboxyl functionalized mannan(Man-COOH),thereby reducing the surface potential of the complex and increasing the circulating time in vivo.Meanwhile,Man-COOH can be specifically recognized by mannose receptors on the surface of macrophages,by which the uptake of the complexes by macrophages was enhanced;thus can improve the transfection efficiency of the complexes,by which the treatment of ALI was facilitated.Part I Preparation and characterization of M/BP-S/siRNAObjectives:To synthesize and characterize the polymer BP-S?BP-C?LP and nanocomposite BP-S/siRNA?BP-C/siRNA?LP/siRNA?M/BP-S/siRNA.Methods:Firstly,2,2-dithiodiethanol diacrylate(SSDA)was synthesized.Then,the bioreducible highly branched poly(?-amino ester)s(BP-S),the insensitive polymer BP-C and the linear polymer LP were successfully synthesized by A2+B3+C2 Michael addition approach.Different polymers were mixed with siRNA to form nanocomposite BP-S/siRNA,BP-C/siRNA and LP/siRNA.Man-COOH was mixed with BP-S/siRNA to synthesize nanocomposite M/BP-S/siRNA.SSDA and BP-S were characterized by 400 MR(1H NMR).The polymers and nanocomposites were characterized by gel permeation chromatography(GPC),dynamic light scattering(DLS),agarose gel electrophoresis assay and heparin sodium displacement assay.Results:Characterization of SSDA by 400 MR(1H NMR)demonstrated that the bioreducible monomer containing disulfide bonds was successfully synthesized.Characterizing each polymer by GPC,we demonstrated that the differences in molecular weight among BP-S,BP-C and LP were not significant.Characterization of BP-S by 400 MR(1H NMR)demonstrated that the highly branched structure of BP-S.The bioreducible property of BP-S was demonstrated by GPC.The particle size and surface charge of BP-S/siRNA and M/BP-S/siRNA were characterized by DLS,demonstrating that the structure was more conducive to cell uptake.Through DLS,agarose gel electrophoresis assay and heparin sodium replacement assay,we demonstrated that BP-S/siRNA could efficiently condense siRNA with bioreducible property.Finally,we monitored the particle size of the nanocomposites in the DMEM medium containing 10%FBS,demonstrating that M/BP-S/siRNA had good serum stability.Conclusions:BP-S/siRNA could efficiently condense siRNA with bioreducible property,which was more favorable for the controlled release of siRNA.M/BP-S/siRNA had better serum stability than BP-S/siRNA.Part ? Cytological study of M/BP-S/siRNAObjectives:A series of cytological studies on BP-S/siRNA,BP-C/siRNA,LP/siRNA and M/BP-S/siRNA were performed to verify the intracellular kinetics,cytotoxicity,and in vitro gene-silencing efficiency of various nanocomplexes.Methods:Flow cytometry was used to quantify the uptake of each nanocomposites by RAW 264.7 cells.Flow cytometry was used to quantitatively detect the uptake of M/BP-S/FAM-siRNA and BP-S/FAM-siRNA by RAW 264.7 cells supplemented with mannan.At the same time,MRC-5 cells were used as control cells for quantitative detection.The endosomal escape and the intracellular release of siRNA of each nanocomposite were detected by confocal microscopy.Cytotoxicity of each nanocomposite in RAW 264.7 cells was detected by MTT assay.Extracellular levels of TNF-? outside RAW 264.7 cells was detected by ELISA.TNF-? mRNA expression levels outside RAW 264.7 cells was detected by Quantitative Real-time PCR.Results:The results of flow cytometry indicated that RAW 264.7 cells had higher uptake levels of M/BP-S/FAM-siRNA,BP-S/FAM-siRNA,and BP-C/FAM-siRNA(M/BP-S/FAM-siRNA had the best intake level)than PEI/FAM-siRNA and LP/FAM-siRNA.In the presence of mannan,the uptake levels of M/BP-S/FAM-siRNA were significantly decreased in RAW 264.7 cells,while the uptake levels of BP-S/FAM-siRNA showed little change.The uptake levels of M/BP-S/FAM-siRNA and BP-S/FAM-siRNA were not significantly different in MRC-5 cells.In the presence of mannan,there was almost no significant change in the uptake level of M/BP-S/FAM-siRNA and BP-S/FAM-siRNA in MRC-5 cells.The results of confocal microscopy suggested that M/BP-S/FAM-siRNA is more effective in escaping endosomes than BP-S/FAM-siRNA,and BP-S is more capable of achieving intracellular release of siRNA compared to BP-C.The results of MTT assay indicated that the cytotoxicity of BP-S and LP was less than that of BP-C;the cytotoxicity of BP-S,BP-C and LP was significantly less than that of PEI.As the weight ratio of the polymer/siRNA was gradually increased,the cytotoxicity of each nanocomposite was also gradually increased.The cytotoxicity of M/BP-S/siRNA was the lowest among the nanocomposites,the cytotoxicity of LP/siRNA and BP-S/siRNA was low,the cytotoxicity of BP-C/siRNA was high,and the cytotoxicity of PEI/siRNA was the highest.The results of ELISA indicated that the ability of BP-S/siTNF-a to inhibit the expression of TNF-? protein gradually increased with the increase of the weight ratio of polymer BP-S/siTNF-a.When the weight ratio of the polymer BP-S/siTNF-a was 30,its ability to inhibit the expression level of TNF-a protein was optimal.When the mass ratio of Man-COOH to BP-S/siRNA was 0.3,its ability to inhibit the expression of TNF-a protein was optimal.Among all the nanocomposites,M/BP-S/siTNF-a had the best ability to inhibit the expression of TNF-a protein,which was superior to BP-S/siTNF-a and BP-C/siTNF-a.The ability of BP-S/siTNF-a to inhibit the expression of TNF-a protein ranked second,which was significantly better than BP-C/siTNF-a and LP/siTNF-a.The results of Quantitative Real-time PCR suggested that M/BP-S/siTNF-a exhibited the best ability to reduce the expression of TNF-a mRNA in cells among all the nanocomplexes,which is significantly better than BP-S/siTNF-? and BP-C/siTNF-a.The ability of BP-S/siTNF-a to reduce the expression of TNF-a mRNA in cells ranked second,which was significantly better than BP-C/siTNF-? and LP/siTNF-a.Conclusions:Compared with other nanocomposites(BP-S/siRNA,BP-C/siRNA,LP/siRNA),M/BP-S/siRNA could escape endosomes effectively and realize intracellular release of siRNA better,which also had other advantages including high cell uptake rate,low cytotoxicity and high gene-silencing efficiency.Part III Preparation of mice model with acute lung injuryObjectives:To establish a mouse model of ALI by intratracheal instillation of LPS.Methods:A total of 8 healthy male BALB/c mice aged 6-8 weeks were selected and their body weights were within the range of 18-20 g.All mice were randomly divided into 2 groups(LPS group and normal group),4 mice in each group.The mice of LPS group were induced to ALI using intratracheal instillation of LPS solution(2.5 mg/kg).The mice of normal group was intratracheally instilled with an equal volume of 0.9%sterile saline.After 24 hours,the mice were sacrificed and the lung tissues of the mice were collected.The pathology of lung tissues of the mice in both groups was evaluated.The expression levels of TNF-a in the lung tissue were determined by ELISA.The expression levels of TNF-a mRNA in the lung tissues were determined by Quantitative Real-time PCR.Results:The pathological condition of the lung tissue in mice suggested that the morphology of lung tissues of mice in normal group is generally normal,while that in LPS group showed obvious pathological changes.The pathological score of the lung tissues in LPS group were significantly higher than that in normal group,and the difference was statistically significant(p<0.05).The results of ELISA indicated that the expression level of TNF-a in lung tissues of the mice in LPS group was significantly higher than that in normal group.The results of Quantitative Real-time PCR indicated that the expression level of TNF-a mRNA of lung tissues in LPS group was significantly higher than that in normal group.Conclusions:LPS could significantly induce ALI in mice,and the animal model was successfully established.Part IV The protective effect and mechanism of M/BP-S/siRNA on LPS-induced acute lung injury in miceObjectives:To study the in vivo anti-inflammatory efficiency and biosafety of nanocomposites which were used to treat ALI in mice.Methods:A total of 24 healthy male BALB/c mice aged 6-8 weeks were selected and their body weights were within the range of 18-20 g.All mice were randomly divided into 6 groups:normal group,LPS group,M/BP-S/siNC group,PEI/siTNF-a group,BP-S/siTNF-a group and M/BP-S/siTNF-a group,4 mice in each group.The mice in LPS group were induced to ALI using intratracheal instillation of LPS solution(2.5 mg/kg).The mice in normal group was intratracheally instilled with an equal volume of 0.9%sterile saline.The mice in M/BP-S/siNC group,PEI/siTNF-a group,BP-S/siTNF-a group and M/BP-S/siTNF-a group were intratracheally instilled with a corresponding nanocomposites(both 500 ?g siRNA/kg)solution 2 hours after LPS induction.After 24 hours,the mice were sacrificed and the tissues of the heart,liver,spleen,lung,kidney,the bronchoalveolar lavage fluid(BALF),the arterial blood and venous blood of the mice were collected.Studies were conducted to assess the anti-inflammatory efficiency of nanocomposites in vivo,including the evaluation of TNF-a and IL-6 expression levels of the mice lung tissues by ELISA and immunofluorescence assays,the expression level of TNF-a mRNA of the mice lung tissues determined by Quantitative Real-time PCR,the expression level of MPO of the mice lung tissues determined by colorimetry,the expression levels of TNF-a and IL-6 in mice BALF detected by ELISA,the expression level of total protein in mice BALF determined by BCA protein quantitative analysis,cell counts in the BALF,the pathological evaluation of the lung tissues and the blood gas analysis.Biosafety studies of nanocomposites comprised of routine blood tests and biochemical tests in mice and the pathological evaluation of various organs(heart,liver,spleen,lung,kidney)in mice.Results:The results of ELISA showed that M/BP-S/siTNF-? had the strongest ability to inhibit the expression of TNF-? and IL-6 in the mice lung tissue.BP-S/siTNF-? and PEI/siTNF-? ranked the second and the third,respectively.The ability of M/BP-S/siTNF-?to inhibit the expression of TNF-? and IL-6 in the lung tissues was significantly better than that of BP-S/siTNF-? with a statistically significant difference(p<0.05).The results of immunofluorescence staining showed that M/BP-S/siTNF-? could decrease the expression level of TNF-? mRNA most effectively and downregulate the expression levels of TNF-?and IL-6 in mice model of ALI.The results of Quantitative Real-time PCR showed that M/BP-S/siTNF-? had the strongest ability to reduce the expression of TNF-? mRNA in the mice lung tissues among all the nanocomposites,which was significantly better than BP-S/siTNF-? with a statistically significant difference(p<0.05).The ability of BP-S/siTNF-? to reduce the expression of TNF-? mRNA in the lung tissues of mice ranked second and was significantly better than that of PEI/siTNF-?.The results of MPO assay in the lung tissues showed that the MPO expression level in the lung tissues of mice was significantly decreased after intratracheal instillation of M/BP-S/siTNF-?,which was significantly lower than that of mice in BP-S/siTNF-? group and PEI/siTNF-? group with a statistically significant difference(p<0.05).The results of ELISA showed that M/BP-S/siTNF-? had the strongest ability to inhibit the expression of TNF-? and IL-6 in mice BALF.The second place was BP-S/siTNF-a and the third was PEI/siTNF-a.The ability of M/BP-S/siTNF-? to inhibit the expression of TNF-? and IL-6 in mice BALF was significantly better than that of BP-S/siTNF-? with statistically significant difference(p<0.05).The results of total protein expression level determination and total cell count assay in mice BALF showed that the total protein expression level and total cell number of mice in M/BP-S/siTNF-? group were significantly lower than that in other groups after administration.The therapeutic effect of M/BP-S/siTNF-? was therefore significantly superior over that of BP-S/siTNF-? and PEI/siTNF-?(p<0.05).The pathological evaluation of the mice lung tissues showed that after the administration of M/BP-S/siTNF-?,the lung inflammation was significantly relieved,and the damage of the lung tissues was significantly reduced.Significant pathological changes were still observed in the lung tissues of the BP-S/siTNF-? group and the PEI/siTNF-? group.The pathological scores of the lung tissues in M/BP-S/siTNF-? group were not significantly different from those in normal group,but were significantly smaller than those in BP-S/siTNF-? group and PEI/siTNF-? group with statistically significant difference(p<0.05).The results of the blood gas analysis in the mice showed that after the administration of M/BP-S/siTNF-?,PaO2 increased significantly,PaCO2 decreased significantly,and the pH value gradually approached the normal level.The therapeutic effect of M/BP-S/siTNF-? was therefore significantly better than that of BP-S/siTNF-? and PEI/siTNF-?(p<0.05).The routine blood test and blood biochemical tests showed that the indices of mice in M/BP-S/siTNF-? group were not significantly different from those in normal group,whereas some indices in PEI/siTNF-? group were significantly different from those in normal group.The pathological evaluation of various organs(heart,liver,spleen,lung,kidney)in the mice showed that the morphological structures of heart,liver,spleen,lung and kidney in M/BP-S/siTNF-? group were not significantly different from those in normal group.Conclusions:M/BP-S/siTNF-? could mediate efficient TNF-? mRNA silencing in the lung tissues of mice to effectively alleviate inflammatory symptoms,and was beneficial to anti-inflammatory treatment of ALI in mice.In addition,M/BP-S/siTNF-? also exhibited the advantages of high biosafety and non-obvious organ toxicity. |
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