| Rheumatoid arthritis（RA）and acute lung injury（ALI）seriously affect the quality of life of humans due to the high disability and mortality.It is hard to achieve effective treatments of RA and ALI because of the complex pathogenic mechanism.As an important immune cell,macrophages play an indispensable role in the occurrence and development of chronic or acute inflammatory diseases.Stimulated by pathogenic signals,macrophages in inflammatory lesions are prone to M1-type macrophages polarization,which can secrete abundant pro-inflammatory cytokines and reactive oxygen species（ROS）,while the counterpart M2-type macrophages can secrete anti-inflammatory cytokines.Regulating the balance of pro-inflammatory/anti-inflammatory cytokines secreted by macrophages and eliminating ROS are important targets for the treatment of such inflammatory diseases.At present,chemotherapy is the main treatment strategy for RA and ALI.There are many defects of chemotherapy,such as poor water solubility,low bioavailability,and side effects of drugs,as well as restricted efficacy of single chemotherapy strategy,limiting its clinical application.In contrast,gene therapy can specifically and endogenously regulate M1/M2 type macrophage polarization-related gene expression,thereby controlling the balance of pro-inflammatory/anti-inflammatory cytokines and eliminating ROS.The keypoint of gene therapy is to select a safe and efficient gene delivery vector.The rapid development of nanobiotechnology enables efficient drug/gene delivery and resultant high therapeutic efficacy.Recently,various nanovectors such as lipid compounds（liposomes and lipid nanoparticles,etc.）and polymers（nanogels and micelles,etc.）have been constructed and widely used in chemotherapy drug and gene delivery for RA and ALI therapy.However,there are still great challenges in designing and farbricating novel safe nanosystems with high gene compression and drug loading efficiency and co-loading capacity of multiple therapeutic drugs for efficienct therapy of RA and ALI.Polyamide-amine（PAMAM）dendrmer is widely used in the therapy and diagnosis of various diseases due to their highly branched structure,hydrophobic cavity and abundant terminal functional groups.A large number of terminal amino functional groups can be used not only for functional modification such as conjugating targeting ligand,but also for loading therapeutic genes and chemotherapy drugs,while the internal hydrophobic cavity can be utilized to entrap hydrophobic drugs to achieve targeted combination therapy of diseases.Particularly,the molecular rigidity of PAMAM dendrimer can be enhanced after entrapped with gold nanoparticles inside,allowing it to condense genes more efficiently and improving gene delivery efficiency by more than 100 times.As another important member of the dendrimer family,the phosphorous dendrimers with a core of hexachlorocyclotrichosphoronitrile（HCCP）have an excellent intrinsic molecular rigidity and unique biological properties due to the enrichment of phosphorus elements in internal branching units and on terminal groups,resulting in its wide use in nanomedicine applications.Furthermore,hydrophobic alkyl long chains can be introduced into the HCCP core to synthesize amphiphilic phosphorous dendron with hydrophobic core and hydrophilic terminal functional groups,which endows it the ability to form micelles in aqueous solution by self-assembly,enhanceing the drug loading capacity in the hydrophobic cavity.Although PAMAM dendrimers and phosphorous dendrimers have good drug loading capacity（chemotherapy drugs and therapeutic genes）,there are few reports about that dendrimers can be employed to co-deliver two or more drugs to regulate macrophages for the anti-inflammatory and/or antioxidative therapy of RA and ALI.Moreover,there are also few reports about the drug effect of phosphorous dendrimers and synergistic anti-inflammation and antioxidation of other drugs,as well as the drug effect and loading capacity of the asymmetric phosphorous dendrimers.Therefore,from the perspective of anti-inflammatory and antioxidative regulation of M1-type macrophages,in this thesis we constructed several dendrimer/dendron based nanomedicine systems for efficient drug/gene delivery to macrophages.The anti-inflammatory and antioxidative therapeutic efficacy induced by chemotherapy,gene therapy and combination therapy of RA and ALI have been investigated.The findings from the results will provide novel ideas and theoretical basis to design and develop effective nanosystem for regulating macrophage polarization.The main research contents are as follows:（1）Multifunctional PAMAM dendrimers entrapped with gold nanoparticles load TNF-αsi RNA for anti-inflammatory and antioxidative therapy of RAThe chemotherapy and gene therapy of RA is limited by the defects such as severe side effects and low availability of drugs.It is difficult to reverse the pro-inflammatory activity of macrophages only using single anti-inflammatory or antioxidative treatment strategy.Functionalized nanovectors can be used to improve drug availability,achieve targeted drug delivery and multi-drug co-delivery,and regulate the secretion of inflammatory cytokines in macrophages at multiple levels for RA therapy.In chapter two,the fifth generation PAMAM dendrimer（G5）was modified with 1,3-propane sultone（1,3-PS）,folic acid（FA）and tocopherol succinate（α-TOS）on its surface,and then the multifunctional dendrimer was used as template to entrap gold nanoparticles to obtain functionalized dendrimer nanovector({（Au0）25-G5.NH2-1,3-PS-（PEG-FA）-（PEG-TOS）}DENPs,named L1),followed by loading with anti-inflammatory TNF-αsi RNA to generate the final nanodrug（L1/TNF-αsi RNA polyplexes）for anti-inflammatory and antioxidative therapy of RA.The in vitro cytotoxicity,cell uptake,cell gene transfection results showed that surface modification of 1,3-PS and targeting ligand FA endowed L1 with an ability of targeted serum-enhanced gene delivery to macrophages,and antioxidativeα-TOS conjuation could improve the water solubility ofα-TOS and resultantly enhance bioavailability.The in vivo micro-CT imaging and hematoxylin-eosin staining（H&E）analysis of joint tissue sections indicated that the anti-inflammatory effect is in an order ofα-TOS<L1<L1/TNF-αsi RNA,andα-TOS modified L1 had a better anti-inflammatory effect than freeα-TOS.After further loading gene TNF-αsi RNA,the prepared L1/TNF-αsi RNA polyplexes had a higher therapeutic efficacy on RA due to the combination of antioxidative and anti-inflammatory therapy.（2）Protonated pyrrolidine modified phosphorous dendrons co-deliver micro RNA mimic and micro RNA inhibitor for ALI therapyThere are many pathways to regulate the transformation of M1-type macrophages into M2-type macrophages,and the effect of single regulatory strategy is limited.Gene therapy can be used to regulate the secretion of inflammatory cytokines of alveolar macrophages for efficient ALI therapy because it can specifically and endogenously regulate the macrophage polarization related genes.Compared with PAMAM dendrimers which possess good molecular flexibility,phosphorous dendrimers with better molecular rigidity can perform higher gene delivery efficiency.In chapter three,a hydrophobic alkyl long chain（C12）was introduced to the HCCP core of first generation of protonated pyrrolidine modified phosphorus dendrimer（G1）,forming the protonated pyrrolidine modified first generation phosphorus dendron（C12G1）,which can self-assemble to format micelle in aqueous solution.Firstly,plasmid DNA encoding enhanced green fluorescent protein（EGFP-p DNA）was employed to compare the gene transfection efficiency between G1 and C12G1 in alveolar macrophages.The experimental results indicated that compared with G1/EGFP-p DNA polyplexes,C12G1/EGFP-p DNA polyplexes can exhibit higher gene transfection efficiency at a low N/P ratio and their optimal gene transfection efficiency is 1.6 times as high as that of G1/EGFP-p DNA polyplexes.Based on this,the C12G1 nanomicelles were utilized to co-load micro RNA 146a mimic and micro RNA 429 inhibitor to obtain C12G1/mi R-mixture polyplexes,which can simultaneously regulate nuclear transcription factor NF-κB and mitogen-activated protein kinase pathway to prevent M1-type alveolar macrophages from polarizing.The results of in vitro cytotoxicity,cell uptake and M1-type macrophage related cytokine assays showed that,compared with single gene therapy,C12G1/mi R-mixture polyplexes treatment can inhibit pro-inflammatory cytokine secretion by M1-type alveolar macrophages more effectively.In vivo micro-CT imaging and H&E analysis of lung tissue slices also demonstated that,compared with single gene therapy,dual gene therapy induced by C12G1/mi R-mixture can enhance the regulation of alveolar macrophages and resultant anti-inflammatory therapy of ALI.（3）TBP modified phosphorous dendron entrapped curcumin（Cur）for anti-inflammatory and antioxidative therapy of ALI.Combination of anti-inflammation regulation of macrophages and ROS elimination is another effective strategy for ALI therapy.As reported,polyphenol Cur with readily oxidized carbon-carbon double bonds and phenolic hydroxyl groups can be used for eliminating ROS,while TBP modified phosphorous dendrimer also can regulate anti-inflammatory effect of macrophages.In chapter four,a hydrophobic alkyl long chain（C11）was introduced into the HCCP core of the third generation of phosphorous dendrimer（G3）,and TBP groups were then modified on its surface,forming the phosphorous dendron nanomicelle C11G3-TBP through self-assembly.The internal hydrophobic cavity of C11G3-TBP nanomicelles can be utilized to entrap hydrophobic Cur to prepare C11G3-TBP@Cur with drug upload efficiency of 21.24%and encapsulation efficiency of 96.86%,for anti-inflammatory and antioxidative therapy application of ALI.The results of in vitro cytotoxicity,cell uptake,ROS level,and M1/M2 phenotype macrophage polarization related cytokine assays indicated that C11G3-TBP nanomicelles possess anti-inflammatory effect and C11G3-TBP@Cur could eliminate ROS and promote the transformation of alveolar macrophages from M1-type to M2-type,enhancing anti-inflammatory and antioxidative therapy of ALI.The Cur entrapment by C11G3-TBP nanomicelles could improve the water solubility of Cur,and resultantly enhance bioavailability.In vivo micro-CT imaging and H&E analysis of lung tissue sections also showed that,compared with single drug therapy of C11G3-TBP or Cur,dual drug therapy induced by C11G3-TBP@Cur nanomedicine can perform an enhanced anti-inflammation and antioxidation of ALI with high efficacy.（4）Synthesis of asymmetric phosphite sodium salt modified phosphorous dendrimer and its delivery of protein drugs for anti-inflammation application.The therapeutic efficacy of protein drug on RA is limited by the low availability.Furthermore,it is difficult to deal with the complex pathogenesis of RA only using single macrophage regulation strategy.Literatrure shows phosphite sodium salt modified phosphorous dendrimers intrinsically posses an ablity to regulate the anti-inflammation of macrophages.However,the relationship between molecular symmetry and anti-inflammatory effect of phosphorous dendrimerhas not been reported.In chapter five,the anti-inflammatory effect on macrophages was compared between 0.5generation phosphite sodium salt modified phosphorous dendrimer AK38 and asymmetric phosphite sodium salt modified phosphorous dendrimers LJ3.Subsequently,AK38 and LJ3 were employed to load bromelain（bro）to obtain AK38/bro and LJ3/bro complexes for regulating anti-inflammation of macrophages and preventing synovial cell activation,thereby achieving the combination treatment of RA.The results of in vitro cytotoxicity,cell uptake,and M1/M2 phenotype macrophage polarization related cytokine assays indicated that,there is no significant difference between AK38 and LJ3 in anti-inflammatory effect of macrophages,while compared with AK38/bro complexes,it is easier for LJ3/bro complexes to be phagocytized by macrophages,indicating better bioavailability of bro.In summary,the efficient drug delivery systems of multifunctional dendrimer/dendron nanoplatform have shown great advantages in regulating anti-inflammation and antioxidation of macrophages.It is of great significance for developing nanomedicine in personalized RA and ALI therapy research. |
PDF Full Text Request