The Construction And Application Of Recognition Molecule-based Nanotheranostic Probes

Cancer is known as ‘The Emperor of All Maladies’,posing major threat to the health of all human being.In most cases,early detection of cancer is the most effective way to improve the survival rate and quality of life.Currently,the clinical diagnostic methods for cancer are detection of cancer bioma rker,medical imaging and biopsy.Due to the drawback of current technology,these methods rarely succeed in early detection of malignant tumors.Meanwhile,molecular imaging is the combination of molecular probes and clinical medical imaging methods,one of the strategies stands out with the advantage of high sensitivity is fluorescence imaging and has been widely used in cancer diagnosis.Regarding cancer therapy,chemotherapy is one of the traditional methods,chemotherapy drugs are organic small molecul es,which usually have problems such as instability and short half-life in blood circulation.To address these issues,nanomaterials have been applied to construct drug carriers,and have successfully prolonged blood circulation time,and achieved passive targeting to tumor taking advantage of the enhanced permeability and retention(EPR)effect.While solving the pharmacokinetics problems of small molecule drugs,nano-drugs are facing new challenges during the process of in-vivo drug delivery.These obstacles include sequestration by mononuclear phagocyte system,intratuomral pressure and nanoparticle extravasation,cellular membrane traversal and subsequent endosomal compartmentalization.Each step is reducing the dose of drug delivered,and the final drug delivery efficiency is the product of each step in the middle.Only when a series of biological barriers to drug delivery should be continuously overcome can the clinical transformation of nanomedicines be truly realized.Besides traditional treatment methods,immunotherapy has been recognized as the fourth treatment of cancer,bringing a new hope for humans to overcome cancer.Cancer immunotherapy is taking advantage of the immune system to attack cancer cells.Immune checkpoint,such as PD-1 and PD-L1,serve as the ‘break’ of immune response.Normally,PD-1 and PD-L1 are only expressed on immune cells to prevent autoimmunity caused by excessive immune responses.Unfortunately,cancers have been taking advantage of this mechanism,they can release immunosu ppressive signals to surrounding T cells by up-regulating their PD-L1 expression,leading to immune escape.In terms of diagnosis,scientists have found lung cancer,melanoma,and kidney cancer cells expression PD-L1 protein,therefore,PD-L1 can be used as a cancer marker for early diagnosis.In terms of treatment,checkpoint inhibitors competitively bind to immune checkpoint to block the interaction between PD-1 on T cells and PD-L1 on cancer cells and restore T cell function of killig cancer cells.At present,clinically used checkpoint inhibitors are monoclonal antibody drugs,which poccess the disadvantages of poor stability and tissue penetration high cost and batch-to-batch variation.As ‘chemical antibody’,aptamer can be the solution of these problems.Aptamers are single-stranded oligonucleotides selected by the systemic evolutionary enrichment of ligands(Systematic Evolution of Ligands by Exponential Enrichment,SELEX).Aptamers bind to their targets by conformational change through hydrogen bonding,electrostatic interaction with Van der Waals force,or π-πstacking.Compared with antibodies,aptamers have the advantages of board target range,small molecular weight,low immunogenicity,large tissue penetration,high stability,easy synthesis and modification.T herefore,aptamers are increasingly used in the diagnosis and treatment of cancer.In order to overcome current limitation of cancer diagnosis and treatment,in this thesis,we focus on the the advances and bottlenecks of nano-carriers to improving the efficacy of chemotherapy,as well as make use of aptamers in the field of early diagnosis and immunotherapy.The main content can be briefly introduced as follows:(1)To overcome a series of biological barriers faced by nanocarriers during delivery in vivo and promote the clinical transformation of nanomedicine,in Chapter2,we constructed a multi-stage delivery nano-carrier to overcome sequential biological barrier during in-vivo drug delivery.Through the host-guest interaction,the drug carrier PAMAM,β-cyclodextrin linear polymer and CD47 peptide are self-assembled into nanoparticles s NDF-CD47,by controlling the proportion of components,the size of assembled nanoparticle can be adjusted.During the blood circulation,nano-carrier can avoid the macrophage clearance taking advantage of the size and CD47 self peptide.After reaching tumor site by EPR effect,the nanoparticle will be disassembled by MMP-2 protease,thereby increasing tumor penetration.Afterwards,nano-carries will enter cancer cell through macropinocytosis,and NLS peptide will lead the carrier to nucleus.s NDF-CD47 has the potential to be applied in the treatment of cancer,and develop a nano-drug platform that can overcome multiple biological barriers.(2)In order to apply the multi-stage drug delivery system s NDF-CD47 for the treatment of cancer,in Chapter 3,we use the DOX as a model drug,DOX was loaed into the hydrophobic cavity of PAMAM-pep Ads,and forming DOX / s NDF-CD47 after assembly.The distribution of the drug in tumor tissues and cancer cells was verified,and the efficacy of the drug was examined in the cellular and in-vivo experiments.Results showed that DOX / s NDF-CD47 have chemosensitizing effect on cancer cells,proving the potenial of this drug to promote the clinical t ransformation of nanomedicines.(3)Compared with chemotherapy,immunotherapy is considered a more promising method to overcome cancer.As an immune checkpoint,PD-L1 protein is an important biomarker in the diagnosis,prognosis and treatment guidance of c ancer.In Chapter 4,we identified a aptamer,Ap3,that can specifically target PD-L1 protein.We examined the binding ability of the Ap3 towards PD-L1 protein at protein and cell levels,respectively,and targeted tumor imaging in animal model.This aptam er can not only become an early diagnosis tool for cancer,but also provide a reference index for PD-L1-related immune checkpoint treatment.(4)Current checkpoint inhibitors are all monoclonal antibody drugs and can be replaced by ‘chemical antibody’,aptamer.In Chapter 5,we combine the advantages of aptamers and the potential of immune checkpoint therapy to construct a bi-specific aptamer simultaneously targeting PD-1 and PD-L1 proteins.The strategy of bi-specific aptamer can constrain the immune acti vation effect at tumor sites and avoid toxic side effects,while reducing the off-target effect.This drug expands the application of aptamers and provides a new tool for immune checkpoint theapy.