Aptamer Served As The Vehicle For Hydrophobic Small Molecules In Tumor Diagnosis And Therapy

Tumor is a major public health problem and remains one of the world’s most destructive diseases.Tumor diagnosis and therapy at molecular level could make a great contribution to patient survival.More than 40%of commercially available drugs are hydrophobic molecules,and with the development of combinatorial chemistry and high-throughput screening technology,more and more drug candidates would be hydrophobic molecules.Aptamers are oligonucleotides obtained by systematic evolution of ligands by exponential enrichment（SELEX）technology which can recognize ions,small molecules,proteins,cells and even tissues specifically.They have been widely used in biosensing,molecular detection and drug delivery,etc.Due to the characteristic of the nucleic acid,aptamers could be synthesized based on chemical methods and show excellent water solubility.Moreover they are also stable in high temperature and organic solvent.Because of the small molecular weight of aptamers,they also show low immunogenicity and strong tissue penetration ability.Herein,aptamers are combined with hydrophobic molecules（contrast agent,drug and photosensitizer）by covalent or non-covalent bond,for improving the solubility and stability of hydrophobic molecules,imaging specific tumor tissue,and killing target cancer cells.Contents in this dissertation include:1.Metastasis from a primary tumor is the leading cause of death for non-hematological cancers.Circulating tumor cells（CTCs）are regarded as the cellular origin of metastasis.Therefore we proposed a"CTC-triggered anti-tumor drug release system":A drug scaffold was placed near main blood vessels during the tumor resection,which was immobilized the photosensitizer-labeled hairpin switch aptamer（HSA）probe.When CTCs flow across the scaffold and were recognized by HSA probe and triggered the structure changing of HSA probe.The HSA probes bound CTCs would flow across the scaffold and circulate in vessels.When they went to vessels near skin and were exposed to the light（i.e.sunlight）,¹O₂ produced by the photosensitizer would destroy CTCs,and thereby achieving the purpose of inhibiting tumor metastasis.The key of this system is that the binding and activating of photosensitizer were not in the same position.The scaffold could be placed in the deep tissue of body to avoid light,photosensitizer could not be activated without CTCs.Meanw hile,the limitations of life time and killing radius of ¹O₂ could also avoid the side effects to non-target cells.Herein,as a proof of concept,we designed a microfluidic device to simulate this process:Part I was made up of a microfluidic chip and could be put in dark place,HSA probes could be immobilized on the surface of the micro-channel to simulate the drug scaffold.Part II was a transparent capillary and irradiated by a household LED to simulate superficial blood vessels.When target cells flow across the microfluidic device,HSA probe could bind them at first,then HSA bound target cells enter the transparent capillary.The photosensitizer on HSA probes would be activated to produce ¹O₂ under irradiation,thereby causing the apoptosis of CTC.Overall,this CTC-specific destruction strategy might provide a novel and promising tool for inhibiting metastasis,tracking the CTCs in circulation system and realizing personalized medicine.2.In order to increase the drug loading capacity and destroy CTCs more effectively,we have established a“sense-and-treat”localized drug delivery system for synergetic destruction of CTCs by employing a DNA Nanodevice based on last work.The DNA Nanodevice was made up of DNA tetrahedron and a HSA probe.The chemotherapy drugs（doxorubicin,Dox）were loaded in edges and photosensitizer was labeled on vertexes of DNA tetrahedron.The DNA Nanodevice showed the capability to promote cellular internalization of anticancer agents,increase drug loading capacity and realiz e synergetic therapy,which enhanced the destructive ability of anticancer agents.As proof of concept,this DNA Nanodevice has the potential to inhibit metastasis by synergetic destruction of CTCs.With the increasing numbers of DNA Nanostructures1-2,more drug delivery systems with higher CTCs destructive ability could be established based on“sense-and-treat”strategy.3.Fluorescence imaging shows advantages of high speed and resolution.Positron emission tomography（PET）has advantages of high sensitivity and strong tissue penetration.Fluorescence contrast imaging nicely complements PET in terms of spatial resolution.Therefore,we developed an aptamer probe for near-infrared fluorescence and PET dual-modality imaging.Firstly,a near-infrared fluorescent dye,Bodipy-636,was synthesized and coupled to the aptamer covalently.After ¹⁸F labeling by exchanging isotope,a near-infrared fluorescence and PET dual-modality imaging probe was achieved.¹⁸F labeling generally carried out in organic solvent to avoid the interference of hydrogen bond.However,DNA cannot dissolve in pure organic solvent.Therefore,the CPG（controlled pore glass）and DNA displacement reaction were employed for ¹⁸F labeling.Meanwhile,these methods also shorted ¹⁸F operator’s exposure time.The aptamer could accumulate in tumor quickly because of its small molecular weight.This[¹⁸F]Bodipy-636 labeled aptamer achieved near-infrared fluorescence and PET dual-modality imaging in specific tumor.Moreover,the[¹⁸F]Bodipy-636 labeled aptamer might overcome the shortage of ¹⁸F labeled antibody,and shows high potential in clinical translation.4.In last three projects,hydrophobic small molecules were coupled to aptamers through covalent bound.We speculated that aptamer bind the hydrophobic small molecule through non-covalent bond based on molecular recognition could also increase the solubility and stability of the hydrophobic small molecule in aqueous.Therefore,we chose docetaxel as a model molecule for hydrophobic drugs.After optimizing the100 mer aptamer（DOC6-100mer）to 22 mer aptamer（DOC6-22mer）,the optimized aptamer maintained high affinity and specificity for docetaxel.The docetaxel-aptamer complex was confirmed by ESI-MS.Meanwhile,the circular dichroism showed the formation of aptamer has been changed after adding docetaxel.Finally,the solubility of docetaxel could be increased by an order of magnitude（14μM to 145μM）in aqueous by adding DOC6-22mer aptamer of docetaxel without the aid of solubilizer.Meanwhile,aptamer did not affect the ability of docetaxel to inhibit cell proliferation.This method of improving the solubility of poorly water soluble drugs by aptamer could also extend to to other hydrophobic small molecule drugs.5.In order to improve the concentration of docetaxel with less aptamer in aqueous based on previous work,we prepared aptamer stabilized docetaxel nanoparticles with microfluidic chip.Sixty aptamer candidates were investigated rapidly by HPLC.11aptamer candidates showed high binding on HPLC were used for ESI-MS assay.6 of them showed aptamer-docetaxel complex on ESI-MS.After optimizing the structure of microfluidic chip,flow rate,and the concentration of aptamer and docetaxel,the nanoparticles with a radius of 89.4±4.9 nm were achieved.The toxicity of nanoparticles against non-drug resistant tumor cells was consistent with free drug.While the toxicit y of nanoparticles was much stronger against drug resistant cell line compared to non-durg resistant tumor cells.The nanoparticles overcome the drug resistant characteristic of drug resistant cell line.Herein,aptamer servered as both a vehicle for docetaxel and a stabilizer for nanoparticles.The microfluidic technology can conveniently increas e the concentration of the hydrophobic drug in the aqueous without the modification of drug.