Construction Of PAMAM-based Immunomodulatory Multifunctional Nanodrug Delivery Systems And Its Applications In Tumor Immunochemotherapy

Malignant tumor is one of the major diseases which seriously threatens the health of human.In recent years,the incidences of cancers still remain at high levels.In clinic,traditional chemotherapy,radiotherapy and surgery are not effective in the treatment of midterm and advanced tumors,thus they could not effectively inhibit the growth and metastasis of malignant tumors.Meanwhile,the physical conditions of patients with advanced tumors are extremely poor,thus the excessive surgery,radiotherapy or chemotherapy usually leads to the severe damages to the immune system,accelerating the metastasis of tumors and the death of patients.Recently,immunotherapy emerges as a promising anti-tumor approach,such as the hot Chimeric antigen receptor T-cell（CAR-NK）therapy and Dendritic cell-activated and cytokine-induced killer cell（DC-CIK）therapy,both showing good prospects in clinical researches.The immune killing of tumors requires the activation of Cancer-immunity cycle.Firstly,tumor cells release antigen-signaling molecules;antigen presenting cell（APC）boosts the antigen processing and presentation;T lymphocytes（T cells）were activated by double signals;T cells migrate,infiltrate,recognize and kill tumors.Under normal circumstances,there are many multiple positive and negative regulatory factors in the Cancer-immunity cycle,thus this cycle is usually stable and difficult to get the positive feedback to eliminate tumor cells.In recent years,chemotherapy has shown the important potentials for activating the Cancer-immunity cycle.Studies indicate that anthracycline anticancer drug doxorubicin（DOX）is an important inducer of immunogenic cell death（ICD）on tumor cells,which could promote the immunogenic cell death of tumor cells（such as melanoma and breast cancer,et al.）for boosting the exposure of damage-associated molecular patterns（DAMPs）,such as calreticulin（CRT）and high mobility group protein B1（HMGB1）.The exposed CRT and released HMGB1 could serve as immune stimulus signals for promoting the recruitment of DC into tumor regions and promoting the engulfment of antigens on dying tumor cells by DC.Subsequently,DC could present antigens to T cells.All of them could promote the positive running of Cancer-immunity cycle.There are many important connections between tumor chemotherapies and immunotherapies.If chemotherapy and immunotherapy are reasonably combined,the tumor treatment efficiency could be efficiently elevated.Although DOX could trigger the immunogenic death of tumor cells,many studies indicate that the immunological effects elicited by DOX are relatively limited,thus it is difficult to kill tumor cells with high efficiency.Therefore,in the first part of this study,in order to efficiently enhance the DOX-elicited anti-tumor immunity,we proposed a combined regimen of immune adjuvant CpG（1826）and DOX.We hoped to use this regimen to achieve potent anti-tumor T cell immunity by using DOX to elicit immunogenic cell death on tumors and utilize CpG to enhance the activities of DC.CpG is a kind of oligodeoxynucleotide（ODN）with immune activation functions and it is also a highly effective immune adjuvant,which is helpful to enhance the differentiation and maturation of DC and promote the presentation of antigens.In order to achieve the effective co-delivery of DOX and CpG,in the first part of this study,a multifunctional nanodrug delivery system basing on PAMAM dendrimer（LMWH/PPD/CpG）was constructed.PAMAM dendrimer is a kind of nanocarrier with regular morphology,stable structure and easy modification character.It has good application prospects in the delivery of chemotherapeutic drugs and nucleic acid drugs.Moreover,there is no report about using PAMAM as the co-delivery vehicle of DOX and CPG.Firstly,DOX was chemically connected on G4 PAMAM dendrimer through the p H-sensitive hydrazone bond,and CpG was electrostatically adsorbed by the positive charges of the amino groups on PAMAM,thus PPD/CpG nanoparticles were fabricated.Although the prepared PPD/CpG nanoparticles have good prospects on promoting the anti-tumor immunity,the PPD/CpG nanoparticles exhibited obvious positive charges.In order to improve the safety of nanocarriers,the anti-metastatic low molecular weight heparin（LMWH）was adsorbed on the surface of PPD/CpG nanoparticles,thus LMWH/PPD/CpG nanoparticles were fabricated.LMWH could effectively inhibit the adhesion effects of platelets to tumor cells,which could also reduce the invasion and metastasis abilities of tumor cells by inhibiting the variation of E-cadherin and N-calcium in the epithelial-mesenchymal transition（EMT）process of tumor cells induced by platelets.Thus,the LMWH/PPD/CpG nanoparticles fabricated in this work simultaneously possessed immune activation and high anti-metastatic capacities.LMWH/PPD/CpG nanoparticles exhibited uniform size distribution,subrotund shape and good serum stability.The cytotoxicity experiment results indicated that the adsorption of LMWH significantly improved the safety of blank carrier.Anti-metastatic mechanisms experimental results indicated that both free LMWH and the designed nanoparticles could effectively regulate the actin cytoskeleton of B16F10 cells,inhibit the adhesion of platelets to B16F10 cells and inhibit the platelets-induced down-regulation of E-cadherin or increase of N-cadherin,showing good anti-tumor metastasis potentials.The immunoregulatory functions of LMWH/PPD/CpG nanoparticles were inspected.The experimental results showed that this fabricated nano-system exhibited the strongest ability to promote CRT exposure and HMGB1 release in vivo.After the therapy of LMWH/PPD/CpG,the ratios of CD40⁺CD11c⁺,CD80⁺CD11c⁺and CD86⁺CD11c⁺cells in draining lymph nodes of tumor-bearing mice were significantly elevated,indicating that LMWH/PPD/CpG could effectively promote the maturation of DC.In addition,when compared to the single-loaded DOX group,the maturation of DC in the co-loaded group of DOX and CpG was significantly enhanced,indicating that CpG could promote the maturation of DC.The infiltration ratios of CD4⁺and CD8⁺T cells in mice tumor tissues were detected,which were both significantly increased,reaching the levels of 2.82 folds or 4.72 folds of HEPES group,respectively.Next,interferon-γ（IFN-γ）in mice serum was also elevated to the highest level among all groups.These above experimental results suggested that LMWH/PPD/CpG nanoparticles possessed the strongest ability to boost anti-tumor immunity.Therefore,LMWH/PPD/CpG nanoparticles effectively inhibited the growth of melanoma tumors and effectively inhibited the lung metastasis of tumors through the combined immune killing effects and the LMWH′anti-metastatic effects.These above results proved that the designed LMWH/PPD/CpG nanoparticles could effectively activate anti-tumor T cell immunity and achieve enhanced tumor immunochemotherapy.However,recent studies have shown that the immune effects mediated by T cells would be severely astricted by the immunosuppressive cells（Marrow-derived suppressor cells,MDSCs）abundantly distributed in the tumor microenvironment,thus leading to the failure of T cells on exerting anti-tumor functions.Therefore,reducing the infiltration of MDSCs in tumor microenvironment is of great significance to enhance the DOX-elicited anti-tumor immunity.Moreover,investigating the influences of MDSCs on immunotherapy is of great values to improve the efficiency of tumor immunochemotherapies.So,how could we reduce the infiltration of MDSCs in tumor microenvironment for enhancing the DOX-mediated immune effects?In order to realize the enhanced immune escape,tumor cells usually promote the secretion of cytokines such as granulocyte colony stimulating factor（G-CSF）and granulocyte-macrophage colony stimulating factor（GM-CSF）,thus greatly boosting the recruitment of MDSCs from bone marrow into tumor microenvironment.Therefore,decreasing the secretion of G-CSF and GM-CSF cytokines could effectively reduce the recruitment of MDSCs into tumors.Studies showed that the tumor cells with overexpressed lactate dehydrogenase A（LDHA）usually secreted more G-CSF and GM-CSF cytokines,thus promoting tumor immune escape.Thus,silencing of LDHA emerges as a potential strategy to weaken tumor immune suppression mediated by MDSCs.In the second part of this research,the co-delivery research of chemotherapeutic drug DOX and small interfering RNA（si RNA）of LDHA（si L）was further conducted basing on the first part of the research,which using DOX to trigger the immune effects,along with using si L to relieve tumor immunosuppression for investigating the combined impacts on anti-tumor immunity.Meanwhile,some important improvements were also conducted on the nanocarriers in the first part,thus a PAMAM-based redox-responsive multifunctional nano-system was fabricated（R-m PDV/PDV/DOX/si L）.In the first part of the study,LWMH/PPD/CpG nanoparticles could only accumulate in tumor sites relying on the passive targeting effects,thus the specificity was weak.So,c（RGDfk）peptide（RGD peptide）was connected on the surface of nanoparticles in the second part by us to endow nanoparticles the active targeting ability.Moreover,the release of nucleic acid in many traditional gene vectors is relatively tardy.Therefore,R-m PDV/PDV/DOX/si L was designed as a redox-responsive nano-system,which could be disintegrated under the cellular abundant glutathione（GSH）,promoting the efficient release of drugs.Firstly,hydrophobic polyester material Poly（δ-valerolactone）（called PVL）was used as the hydrophobic segment.And the redox-responsive compound3,3’-dithiodipropionic acid（DA）which contained disulfide bond was used as the linker to connect G2 PAMAM dendrimer or methoxy-polyethylene glycol-amino（m PEG-NH₂）,thus two kinds of amphiphilic polymers were obtained.In order to conjugate RGD peptide,the m PEG-NH₂ on the amphiphilic polymers was replaced by maleimide-polyethylene glycol-amino（mal-PEG-NH₂）and RGD was conjugated through the reaction between maleimide and the sulfhydryl of RGD.R-m PDV/PDV/DOX/si L nanoparticles were fabricated by the self-assembly of these three obtained amphiphilic polymers,with DOX encapsulated in the core and si L condensed by the positive charges of nanoparticles.R-mPDV/PDV/DOX/siL nanoparticles exhibited uniform size distribution,good serum stability and the redox-responsive release ability of DOX or si L.The results of in-vivo targeting assay suggested the fluorescence intensity of the RGD-modified nanoparticles in tumor region was significantly stronger than that of the ligand-free nanoparticles,indicating that RGD could effectively promote the tumor accumulation of nanoparticles.Next,the immune regulation effects of R-m PDV/PDV/DOX/si L were investigated.After the silencing of LDHA by si L,the secretion levels of G-CSF and GM-CSF of tumor cells were significantly reduced,the infiltration level of MDSCs in tumor microenvironment and the secretion level of immunosuppressive factor interleukin-10（IL-10）were also significantly reduced.Subsequently,the ratios of CD4⁺T cells and CD8⁺T cells in the tumor tissues were greatly elevated by R-m PDV/PDV/DOX/si L nanoparticles.The levels of IFN-γand TNF-αin mice serum were also significantly increased,while the level of the immunosuppressive factor IL-10 was significantly decreased.The results indicated that R-m PDV/PDV/DOX/si L nanoparticles possessed effective functions on relieving tumor immunosuppression and enhancing the anti-tumor immunity.Therefore,R-m PDV/PDV/DOX/si L nanoparticles effectively inhibited the growth of tumor（inhibition rate was 85.41±0.07%）and the lung metastasis of tumor.Then,preliminary safety investigation was investigated.The results in the staining of tumor sections and the detection of serum biochemical indexes indicated that R-m PDV/PDV/DOX/si L had no obvious toxicity to the major organs of mice.Therefore,the R-m PDV/PDV/DOX/si L nanoparticle designed in this study was an ideal tumor-targeting formulation with good safety.In summary,this study constructed several new types of multifunctional nanodrug delivery systems basing on the potential tumor immunochemotherapy strategies.Thus,the tumor targeted co-delivery of chemotherapeutic drugs and nucleic acid drugs was realized,the aim of effect-enhancing and toxicity-reducing was also achieved,revealing more possibilities for the high-efficient treatments of malignant tumors.