Construction Of Poly (Amidoamine) Dendrimer-Based Functional Nanomedicines For Photothermal/Immune Antitumor Therapy

Cancer remains one of the major diseases that threaten human health.Traditional cancer treatments of surgery,chemotherapy and radiotherapy face challenges such as incomplete elimination of cancer cells,easy metastasis and recurrence,and side effects on normal tissues.Cancer is essentially a genetic mutation in the body that causes healthy cells to transform into cancer cells and further evolve into malignancy.Tumor tissue,with an immunosuppressive tumor microenvironment,differs from normal tissues in many aspects including growth and metabolism patterns,microenvironmental structure and matrix composition.In recent years,based on these differences,various novel tumor treatment strategies with minimal invasiveness and toxic risks have been developed,including photothermal therapy（PTT）,gene therapy and immunotherapy.PTT enables efficient cancer cell killing and immunogenic tumor death（ICD）generation by virtue of local tumor temperature rise,but it may cause residual tumor recurrence and metastasis due to unsatisfactory tumor tissue penetration during treatments.Gene therapy repairs the abnormality of tumor at the root through correction of specific tumor-related gene mutations.In the meantime,immunotherapy activates the patient’own immune cells to specifically attack cancer cells,allowing the formation of immune memory protection.Toward the purpose of efficient tumor treatment,the killing of tumor cells,activation of immune cells and/or repair of mutated genes in tumor cell（or immune cell）should be major principles taken into consideration,which simultaneously contribute to enhanced therapeutic outcome and long-lasting tumor suppression effects.The according key technology is to rationally design multifunctional nanocarrier systems to integrate different therapeutic modalities,relying on the latest progress in nanotechnology and nanomedicine.Among various nanocarriers,dendrimer has received wide attention due to its unique physical and chemical properties.It is a class of highly branched,synthetic and monodisperse macromolecules with very precise nuclei,internal spaces,as well as large number surface groups that can be easily functionalized.Among them,poly（amidoamine）（PAMAM）dendrimer represents the most widely studied dendrimer type,with internal cavity and surface groups that can be physically or chemically-coupled with one or multiple contrast and/or therapeutic agents,allowing efficient delivery to tumor sites for diagnosis,therapy or theranostics after functionalization（e.g.,with targeting and anti-protein absorption reagents）.For example,the polycationic nature of PAMAM dendrimer renders it with excellent gene compression ability,and especially,entrapping gold nanoparticles（NPs）in the internal cavity helps to maintain its molecular rigidity,which further improves its gene delivery efficiency by more than 100-fold.In addition,PAMAM dendrimers after the grafting of targeting reagents such as folic acid or RGD peptide confer targeting specificity to cancer cells with high expression of relevant receptors,and zwitterionic modification greatly enhance the anti-protein absorption ability of dendrimers during blood circulation.Dendrimer-based nanocarriers and nanomedicines still face problems in tumor treatment applications,including the following aspects:（1）Single therapeutic approach often fails to inhibit tumor migration and invasion,and thereby the combination of different modalities is needed;（2）How to use the good gene compression property of dendrimer in combination with immune checkpoint blockade for targeted immune cell modification,and further enhance anti-tumor immune response for long-term immune memory protection;（3）During PTT treatment,tumor cells remove damaged organelles or harmful substances produced by PTT through cooperative autophagy and evade immune surveillance,which constrains the PTT efficacy and the consequential immune responses.To address these issues,a series of dendrimer nanodrug platforms were constructed for combination PTT/immunotherapy against tumors to implement potent and long-lasting tumor treatment.The main contents of this thesis are as follows:（1）Zwitterionic dendrimer-entrapped CuS NPs for PA imaging-guided combination PTT/gene therapy of tumorsPTT has received widespread attention owing to its minimal invasiveness and high local killing efficiency,but single-modal PTT is limited in achieving complete tumor migration and invasion inhibition,and therefore combination therapy is required.In recent years,CuS NPs,as a type of inorganic photothermal reagent,have attracted rising attention in the field of tumor PTT due to their good near-infrared region absorption and excellent photothermal conversion efficiency.In the meantime,tumor hypermethylated gene 1（HIC1）has been reported to play a vital role in tumor metastasis,and the up-regulation of HIC1 expression in tumors by means of gene transfection can effectively inhibit tumor metastasis.In this chapter,we combine PTT-mediated cancer cell killing with gene repair treatment using a dendrimer nanoplatform to simultaneously achieve tumor growth and metastasis suppression.We constructed dendrimer-entrapped CuS NPs（RGD-CuS DENPs）with targeting and anti-protein absorption properties to complex plasmid DNA（p DNA）encoding HIC1 for PA imaging-guided tumor PTT/gene therapy.The results show that the prepared multifunctional RGD-CuS DENPs possess a mean CuS core diameter of 4.2 nm,good colloidal stability,as well as excellent photothermal effect（with a photothermal conversion efficiency of 49.8%）and excellent PA imaging efficacy.Meanwhile,the zwitterionic RGD-CuS DENPs have good cytocompatibility and can be used serum-enhanced gene delivery of p DNA-HIC1.Moreover,RGD-CuS DENPs achieve specific targeting toα_vβ₃ integrin-overexpressing cancer cells due to the surface modification of RGD peptide.In vivo assays demonstrate that the constructed RGD-CuS DENPs/p DNA complexes effectively kill human-derived triple-negative breast cancer cells（MDA-MB-231）and inhibit tumor growth under NIR irradiation.Meanwhile,p DNA-HC1 delivery-mediated gene therapy allows the blockade of cancer cell lung metastasis.（2）Zwitterionic dendrimer-entrapped Au NPs for YTHDF1 gene silencing of DCs to boost tumor immunotherapyDue to the existence of tumor cell immune escape and defective immune cell antigen presentation,it is often hard to arouse abundant anti-tumor immune responses in the body during immunotherapy for satisfactory therapeutic effects,even though large amount of neoantigens can be detected in the cancer patients.Hence,it is rational to apply nanomedicines for targeted modification of the unfavorable factors in DCs and combine with immune checkpoint blockers to enhance the anti-tumor immune response.DCs,as antigen-presenting cells,are responsible for capturing tumor neoantigen and T cell cross-priming to activate the downstream T cell-based immune responses,thereby effectively killing cancer cells.However,high expression of YTHDF1in DCs reduces their tumor antigen cross-presentation ability,resulting in low immune responses and difficulties in complete tumor elimination.In addition,tumor cells overexpress programmed death ligand 1（PD-L1）as an immune checkpoint receptor to programmed death-1（PD-1）on the surface of T cells,which leads to T cell apoptosis and limits the immune response in vivo,causing tumor cell immune escape.In this chapter,in combination with PD-L1 inhibitors of tumor cells,we genetically modify the immune cells through a dendrimer-based nanoplatform to enhance antitumor immunity.We constructed an anti-protein absorption reagent zwitterion 1,3-PS and targeting reagent mannose-modified dendrimer-entrapped Au NPs({（Au⁰）₂₅-G5.NH₂-Man-PS₂₀},for short MDNP)to load YTHDF1 si RNA（si YTH）for genetic engineering of DCs to silence the expression of YTHDF1 protein（an important reader protein responsible for RNA m⁶A methylation）,which promotes DCs maturation and potentially enhances their antigen-presenting ability,restoring T cell killing effect in combination with PD-L1 antibody for efficient tumor immunotherapy.The results indicate that the MDNP displays good colloidal stability and an average Au core diameter of 1.8 nm.In addition,due to the modification of mannose as targeting molecules,it facilitate targeted uptake in DCs.In vitro studies show that MDNP and MDNP/si YTH complexes have excellent cytocompatibility,and MDNP/si YTH complexes transfected DCs show downregulated YTHDF1 gene and protein expression levels,along with enhanced DCs maturation,holding promise for improving their antigen-presenting ability.In vivo studies show that the MDNP/si YTH complexes combined with PD-L1 antibody-based immune checkpoint blocker effectively improves tumor treatment and enhance T cell-based immune responses in spleen and tumor tissues.（3）Dendrimeric photothermal nanodrugs for autophagy inhibition-enhanced tumor PTT/immunotherapyDuring PTT,tumors resist photothermal-induced cell damage and evade immune surveillance through autophagy,resulting in poor immune response triggered by PTT and the companied ICD.In addition,the immunosuppressive tumor microenvironment and inadequate T cell infiltration in tumors limits the efficacy of immunotherapy.The complex tumor growth pattern and immunosuppressive tumor microenvironment has been reported to provide multiple protective mechanisms for tumors.Of these,cancer cells remove damaged organelles or harmful substances during PTT treatment through cooperative autophagy,which helps them to evade immune surveillance and cause constrained PTT treatment efficacy and limited cancer cell ICD generation.Indocyanine green（ICG）is a small-molecular photothermal agent that has been used for fluorescence tracing and PTT.Meanwhile,chloroquine（CQ）is an autophagy inhibitor and immunomodulator,which enables the regulation of tumor autophagy and tumor microenvironment in PTT toward amplified in vivo anti-tumor efficiency.In this chapter,we implement enhanced combination PTT/immunotherapy through tumor autophagy inhibition by integrating the photothermal reagent（ICG）and autophagy inhibitor（CQ）within one dendrimer nanoplatform.The results show that the designed G5.NHAc-ICG/CQ（GIC）nanodrug display excellent stability,biocompatibility and high photothermal conversion efficiency（39.7%）.The in vitro results verify that GIC nanodrug enables CQ-mediated autophagy inhibition for enhanced PTT to kill tumor cells under an 808-nm laser irradiation,inducing cancer cell apoptosis and necrosis.Furthermore,the PTT effect promote cancer cell ICD generation to regulate damage-associated molecular patterns for immune stimulation,promoting DCs maturation and activating T-cell immune responses for tumor immunotherapy.Meanwhile,CQ as an immunomodulator,can induce the activation of NF-κB pathway and reprogram tumor-associated macrophages into anti-tumor M1 phenotype,enhancing the immune responses and improving the immune attacks against tumors.Finally,in vivo experiments show that combination treatment of GIC nanodrug with PD-L1 antibody augment the in vivo immune responses and triggers the production of cytotoxic T cells,which significantly inhibits the growth of both primary and distal tumors in tumor-bearing mice.In conclusion,the integration of PTT/immunotherapy along modification of specific tumoral and immune targets can help to achieve excellent and long-lasting antitumor therapeutic effects.The developed functional dendrimer-based nanoplatforms in this study provoke new insight and feasible approach for combination tumor treatment strategy and may provide guidance for personalized tumor nanomedicine.