Study On Functional Delivery Systems Of Nucleic Acid Therapeutic Agents For Reversal Of Tumor Immunosuppression

As a major disease threatening human health,cancer is hallmarked by its immunosuppression and immune escape.Traditional treatments such as chemotherapy and radiotherapy suffer from the limitations including severe side effects,therapy-induced resistance and unsatisfactory prognosis.By using the body's immune system,immunotherapy holds great promise in cancer treatments due to its less toxic effects,lower recurrence rate and decreased long-term mortality.Due to the immunosuppressive microenvironment in tumor sites,it is of crucial importance to reverse the cancer immunosuppression in cancer immunotherapy.Nucleic acid therapeutic agents can overcome tumor immunosuppression and immune escape via diverse mechanisms.However,nucleic acid drugs are easily degraded by complex physiological environments in blood,tissues and cells.It is of great significance to develop efficient and safe nucleic acid delivery vectors.In this thesis work,a series of safe and efficient gene delivery vectors to deliver nucleic acid therapeutic agents to particular cells were designed and prepared.Their efficiency in activating anti-tumor immunity and reversing immunosuppression were evaluated.The gene vectors are mainly composed of natural polymers,functionalized natural polymer derivatives and calcium carbonate.All components in the delivery systems have ideal biocompatibility and biodegradability.In Chapter 1,the research progress in gene delivery vectors are reviewed,with the emphasis on targeted gene delivery systems.The immunosuppressive factors in tumor microenvironment as well as their reversal strategies are introduced.In the tumor microenvironment,macrophages infiltrated into tumor cells are predominantly polarized to the M2 phenotype with anti-inflammatory functions that facilitate tumor growth.To address this issue,Chapter 2 focuses a macrophage targeting delivery system for efficiently delivery of Cp G oligodeoxynucleotides(ODN)to activate macrophages and induce anti-tumor immune responses in cancer immunotherapy.Mannosylatedcarboxymethylchitosan/protaminesulfate/Ca CO₃/ODN(MCMC/PS/Ca CO₃/ODN)nanoparticles were prepared using a facile self-assembly method.The functional components,including MCMC to endow the nanoparticles with macrophage targeting ability,PS to improve the ODN loading capacity and enhance the cell uptake,and Ca CO₃ to encapsulate ODN and induce the favorable p H sensitivity,were introduced to the delivery systems by self-assembly.The effects of ODN delivery on immune cells were studied in RAW 264.7 cells.Due to the mannose mediated endocytosis,MCMC/PS/Ca CO₃/ODN nanoparticles exhibit a higher ODN delivery efficiency and a significantly enhanced immune stimulation capacity as compared with CMC/PS/Ca CO₃/ODN nanoparticles.The regulation of NF-?B activity by our ODN delivery system results in dramatically increased production of proinflammatory cytokines including IL-12,IL-6,and TNF-?in RAW 264.7 cells.The significantly increased CD80 expression after stimulation by the ODN delivery systems indicates the successful modulation of the macrophage polarity to the anti-tumor M1 phenotype.The multifunctional macrophage targeting delivery system developed has promising applications in delivery of Cp G ODN in cancer immunotherapy.Chapter 3 is concentrated on a dual-targeting vector for delivery of Cp G oligodeoxynucleotides(ODN)to macrophages to overcome cancer-associated immunosuppression.The mannosylated carboxymethyl chitosan(MCMC)and hyaluronic acid(HA)were introduced simultaneously for macrophage targeting.The positively charged nanocore was formed by protamine sulfate(PS)for ODN complexation,and then the targeting components were assembled into the PS/ODN nanocore via electrostatic interactions to obtain dual-targeting nanoparticles(MCMC/HA/PS/ODN).The effects of ODN delivery on immune cells was studied in J774A.1 cells.Compared to mono-targeting or untargeting nanoparticles,dual-targeting nanoparticles exhibit enhanced delivery efficiency and immune stimulatory activity,resulting in dramatically enhanced secretion of pro-inflammatory cytokines and a successful shift to the M1 macrophage phenotype.Besides macrophages,the influence of these delivery systems on tumorous MCF-7 cells was also investigated.In MCF-7 cells,the increased expressions of NF-?B,PIK3R3,p-AKT caused by activated NF-?B and PI3K/AKT signalings were observed.Nevertheless,up-regulated Fas as well as Fas-L may induce Fas/Fas L-mediated apoptosis.The effect of Cp G ODN on cancerous cells is pleiotropic.Upon ODN stimulation,the up-regulated proteins may have contradictory effects on cell growth and proliferation.As an overall result,ODN delivery systems do not show apparent effects on the viability of MCF-7 cells.This study highlights the importance of targeting delivery of Cp G ODN to macrophages.As reported in Chapter 4,to further enhance the efficiency in modulating the macrophage polarity to the anti-tumor M1 phenotype,a fusion peptide functionalized gene delivery system with macrophage and tumor targeting ability was prepared to deliver the plasmid DNA encoding IL-12 gene(p DNA IL-12)for macrophage re-polarization as well as reversal of cancer immunosuppression.A fusion peptide containing tuftsin sequence which can interact with Fc receptors in macrophages and neuropilin-1 in tumor cells,and hyaluronic acid(HA)which can interact with CD44 overexpressed in both macrophages and tumor cells were introduced to the delivery system by self-assembly to form peptide/hyaluronic acid/protamine/Ca CO₃/DNA nanoparticles(PHNP)with macrophage targeting and tumor targeting capabilities.PHNP provides an efficient immunoregulation on J774A.1 cells to shift the anti-inflammatory M2 phenotype to the anti-tumor M1 phenotype with enhanced secretion of pro-inflammatory cytokines and increased expression of M1 markers.Owing to the improved delivery efficiency caused by the fusion peptide and HA,the transfection mediated by multi-functional PHNP can up-regulate IL-12 as well as down-regulate IL-10 and IL-4 more effectively as compared with the nanoparticles without HA and/or peptide decoration.More importantly,the gene delivery system can also deliver p DNA IL-12 to targeted cancerous He La cells to realize the secretion of IL-12.PHNP not only enables tumorous cells to produce IL-12,but also down-regulates CD47 and up-regulates CD80 and HLA-1 in the malignant cells,indicating the gene delivery system can effectively reverse tumor induced immunosuppression.The immunosuppression in tumor microenvironments is induced by tumor malignization and progression.To ultimately reverse tumor-induced immunosuppression and immune escape,the target of delivery systems is shifted from immune cells to tumor cells from Chapter 5.Since Wnt/?-catenin pathway is involved in the expression of PD-L1 in tumor cells.In Chapter 5,a multifunctional delivery system for efficient delivery of CRISPR-Cas9 plasmids to targeted cancerous cell nuclei and knock out the CTNNB1gene encoding?-catenin is reported.In the delivery system,AS1411 aptamer functionalized hyaluronic acid(AHA)and TAT-NLS fusion peptide functionalized hyaluronic acid(PHA)were introduced the the surface of Ca CO₃/PS/Plasmid to obtain PHA/AHA/Ca CO₃/PS/Plasmid through self-assembly.The AS1411 mediated tumor cell/nuclear targeting and TAT-NLS induced enhanced endocytosis and nuclear localization result in obviously increased cellular uptake and nuclear transport.As a result,PHA/AHA/Ca CO₃/PS/Plasmid.could mediate more effectively genome editing as compared with other nanoparticles without funtional components,resulting in an efficacious knockout of CTNNB1 gene coding?-catenin and down-regulation of?-catenin.Analysis of Wnt/?-catenin pathway activity reveals that down-regulation of?-catenin can effectively inhibit Wnt/?-catenin pathway activity and significantly down-regulate downstream protein expression,such as cyclin-D1,VEGF,Bcl-2,c-myc and PD-L1.After knocking out the CTNNB1 gene,cancerous H1299 cells exhibit increased proportion of cell apoptosis.More importantly,the edited H1299 cells show a markedly supressed PD-L1/PD-1 binding,and markedly enhanced T cell-mediated killing.The above results demonstrate that the prepared multifunctional nanoparticles for genome editing can effectively reverse tumor immune escape through relieving PD-L1/PD-1mediated T-cell dysfunction.In addition to tumor immunosuppression,Wnt/?-catenin pathway is also involved in the regulation of other tumor cell behaviors,including tumor growth,migration and invasion,and cancer stemness.Chapter 6 is focused on a multifunctional delivery system for delivery of genome editing plasmid targeting?-catenin to re-modulate cancer cell properties.In the delivery system,the CRISPR-Cas9 plasmid is combined with protamine with membrane and nuclear translocating activities and co-precipitated with Ca CO₃,which is further decorated by AS1411 functionalized carboxymethyl chitosan(ACMC)and TAT peptide functionalized carboxymethyl chitosan(TCMC).The AS1411 mediated tumor cell/nuclear targeting and TAT induced enhanced endocytosis result in obviously increased cellular uptake and nuclear transport.As a result,the CRISPR-Cas9 plasmid can be efficiently delivered to cancer cell nuclei to mediate genome editing,resulting in an efficacious knockout of CTNNB1 gene coding?-catenin.More importantly,down-regulation of?-catenin could effectively prevent its enrichment in nucleus and then significantly down-regulate the expression of proteins,such as vimentin,Snail,MMP-2,MMP-9,CD44,Nanog,and Oct4 to prevent tumor progression and metastasis.The edited cancerous cells exhibit favorable re-modulated properties including inhibited growth,suppressed migration and invasion,and reduced cancer stemness.