The Study of Multifuntional Upconversion Nanoparticles Based on Aptamers

Author:Wu Yuan

Supervisor:tan wei zuo


Degree Year:2018





With the rapid development of life science and modern medical technology,human has made great progress in exploring and realizing early diagnosis and treatment of diseases by using molecular biology and different kinds of dia gnosis and treatment.However,according to data published by the National Cancer Center in2017,the number of people suffering from cancer is rising,and c ancer is still the major cause of death in people.Traditional cancer treatment methods,such as chemotherapy,often cause serious side effects because of non-specific effects of chemotherapy drugs.Common cancer diagnostic techniques,such as X-ray scan or CT scan,have low sensitivity,resulting in false positive signals and excessive diagnosis or treatment,or missing optimal treatment time.Therefore,it is very important to establish and develop highly sensitive and accurate early cancer diagnosis tech niques and efficient targeted cancer treatment to improve the survival rate of cancer patients.Since the concept of the aptamer was first proposed in the 1990s and the research results have been published,aptamers have been widely studied in molecular biology,analytical chemistry,biochemistry,medicine,bioinformatics,pharmacy and nanomaterials after nearly 30 years of research.Aptamers,called“chemist’s antibodies”,are a new type of recognizing molecules with many merits,such as high selectivity,strong binding ability,wide range of targets,low immunogenicity,good biocompatibility,easy synthesis,easy modification and easy preservation.They have become a new type of targeting ligands that can be used for precisely early diagnosis of cancer and efficiently personalized treatment.On the other hand,nanomaterials have broad application prospects in the field of bioimaging and therapeutics due to their many advantages,such as unique optical properties,small size,large surface area,strong loading capacity,good biocompatibility and easy modification.Based on the above research background and aimed at the application of aptamers functionalized nanomaterials and virus vectors to achieve targeted early diagnosis and treatment of cancers,we have developed a series of new multifunctional nano-diagnosis and treatment systems for biological imaging and therapeutic research.Specifically include the following aspects:(1)Traditional photodynamic therapy suffers mainly from insufficient tissue penatration and photosensitizers without targeting capability.Therefore,it is extremely necessary to construct a method with high selectivity and accurate localization to controllably generate singlet oxygen,so as to provide more efficient photodynamic therapy with fewer side effects.In Chapter 2,we constructed aptamer-guided G-quadruplex functionalized upconversion nanoparticles for targeted bioim aging and photodynamic therapy by extending the 5’-end of sgc8 aptamer sequence with a G-rich short sequence to form a G-4 structure,which can load photosensitizer TMPyP4.Then,the 5′-modified thiol of the sequence was covalently cross-linked to the surface of the upconversion nanoparticles through sulfo-SMCC crosslinker.Once the probes were delivered into target cells,the upconversion nanoparticles were excited by near-infrared light to emit visible light to activate the photosensitizer to generat e singlet oxygen,finally cancer cells were killed.(2)It is reqiured to transfer oleic acid-capped hydrophobic upconversion nanoparticles to aqueous phases before using them in biological applications.It is reported that it takes as long as 48 h to use the Lemieux-von Rudloff reagent to convert the hydrophobic upconversion nanoparticles into aqueous phase by the surface ligand oxidation method.Therefore,after studying carefully the oxidation mechanism of the carbon-carbon double bond in the olefin by Lemieux-von Rudloff’s reagents,we envisaged whether it is possible to accelerate the oxidation process by appropriately adjusting the proportions of the KMnO4 and NaIO4 reagents used in the Lemieux-von Rudloff reagents.Moreover,the oxidation reaction is carried out under weak alkaline condition,and the product after oxidation reaction of KMnO4 under alkaline condition is generally manganese dioxide(MnO2).In Chapter 3,we adjusted the amount of KMnO4 and NaIO4 reagents used in the Lemieux-von Rudloff Reagent,and we found some precipitates were grown on the surface of upconversion nanoparticles,and it happened that the fluorescence of upconversion nanoparticles was quenched.At the same time,the hydrophobic nanoparticles were successfully transferred into water phase.Then,a series of characterizations were adopted to identify the surface precipitates as manganese dioxide,which provided a basis for the construction of a new nanodiagnostic probe.(3)Our group has reported the use of manganese dioxide nanosheets for gene silencing therapy,but MnO2 nanosheets were intratumoral injected into athymic nude mice instead of intravenous injections in vivo studies because of the presence of glutathione in the blood.Manganese dioxide will be degraded in advance in the blood circulation,resulting in side effects.In order to protect the manganese dioxide from premature reduction by glutathione in the blood before entering the target cells,in Chapter 4,we constructed a novel targeted nanotherapeutic probe to protect manganese dioxide.The UCNPs@MnO2 developed in Chapter 3 was coated by mesoporous silica for drug loading.The pores of mesoporous silica were blocked with gelatin nanolayers,and the aptamers were finally modified on the surface of gelatin.Once the nanoprobe s were delivered into the target cells,gelatin would be degraded by the acidic environment of the lysosome of the target cell,resulting in t he release of the drug and the entry of GSH,at this time the inside MnO2 would be reduced to Mn2+by glutathione in the target cell,which could be used as a contrast agent for magnetic resonance imaging.At the same time,the fluorescence of upconversion nanoparticles was recovered after the reduction of MnO2,achieving activatable fluorescence imaging.The nanosystem not only can achieve targeted dual-mode imaging and pH-stimulated drug controlled release,but also promotes the development of manganese d ioxide nanosheets in cancer diagnosis and treatment.(4)In addition to photodynamic therapy and chemotherapy,gene therapy also provides a possible treatment for many human diseases.The ideal gene therapy vectors must be safe,efficient,and stable to achieve gene transfection,that is,the vectors must be able to accurately delivered and release the gene.Therefore,it is considerably important to obtain targeted gene transfection vectors for successful gene therapy.In Chapter 5,we successfully constructed targeted adeno-associated type 2 virus(AAV2)vectors for targeted gene transfection based on aptamers.In this work,the aptamers were assembled with DNA dendrimer structure,and then were covalently cross-linked with adeno-associated virus via a disulfide-containing cross-linking agent.Finally,the target gene transfection of the AAV2 vectors carrying the GFP gene was achieved.The multi-effect of DNA dendritic nanostructures increased the affinity of the aptamer to the target cell.In this strategy,AAV2 were chemically modified with aptamers,so that the modified vectors could infect target cells through aptamer-mediated endocytosis,which can solve the problem that AAV2 vector can only infect cells positively expressed with heparan sulfate,finally the application of AAV2 vector in gene therapy can be broadened.