Preparation Of Novel Self-Assembled Biomaterials And Its Application In Cancer Immunotherapy

Due to Ureidopyrimidone（UPy）contains quadruply hydrogen-bonding（QHB）structure,it possesses extremely strong reversibility and dynamic interaction.The UPy units link the molecules containing the UPy by their selfcomplementary quadruple hydrogen bonding ability.This property can be exploited to promote self-assembly and then build polymer structure.UPy units can be synthesized from isocytosine and isocyanate derivatives,UPy-based supramolecular systems have been extensively studied over the past decade.Because polyethylene glycol（PEG）has many excellent properties,such as Non-toxic,non-irritating,good biocompatibility,easily degradable in the bodies and so on,it is widely used in pharmaceutical preparations.The treatment of cancer by strengthening the patient’s immune system is called cancer immunotherapy（CIT）.In recent years,with the continuous development and optimization of cancer immunotherapy,many cancers have undergone a dramatic transformation,particular in melanoma,renal,bladder and lung cancers.Compared with traditional chemotherapy,immunotherapy has better effects to restrict and eliminate tumors.In recent years,immunotherapy is the forefront of anti-cancer therapies.Because the drugs used in cancer immunotherapy,such as monoclonal antibodies,Toll-like receptor agonists,cell penetrating peptides,and cell-targeting peptides,are easily inactivated when administered directly,it is necessary to research some new delivery systems to improve the therapeutic effect.We synthesized amphiphilic polymers by ureidopyrimidone-functionalizing polyethylene glycol.And then we investigated its capacity of encapsulating protein and loading DNA in water,the ability to transport proteins into the cells as a carrier,and the therapeutic effects in animal tumor models.Our works mainly include the following points:1.Preparation of Amphiphilic PolymersBased on 2-amino-4-hydroxy-6-methylpyrimidine,we utilized the special reactivities of amino groups to extend the carbon chains,and modified the ends of carbon chains with alkene or alkyne bonds,then connected monomer molecule and PEG-NH₂ or PEG-N₃ via Micheal addition or"Click"reactions,respectively.2.The studies of biological properties of the polymersWe examined the dispersibility of the material in water,then observed its size and shape.The capacity of loading fluorescent BSA was evaluated by using microplate reader.During this process,we found that the material can self-assemble in water to form regular shaped particles that can encapsulate proteins.Finally,it was verified by agarose gel electrophoresis and polyacrylamide gel electrophoresis that the material can be complexed with ureidopyrimidone-functionalized DNA.3.Cell endocytosis assayAfter loading fluorescent BSA to the materials,we added them into 96-well plates,in which have paved with Raw 264.7 cells.Then observed the endocytosis of cells use an inverted microscope.During this process,we found that the materials possess an outstanding biocompatibility and almost have no toxicity to cells.However,due to the large size of the particles,proteins cannot be delivered into cells by endocytosis of cells.In order to fully take advantage of the many excellent properties of materials,we transformed it into a delivery materials that work in the outside of cells.4.In vivo experimentsWe constructed a melanoma animal model by subcutaneously injecting B16F10cells into C57 black female mices.After the tumor grew up,the PBS solution containing materials,which were loaded therapeutic proteins（anti-PD-1,anti-CTLA-4 antibody）and DNA（CpG）,was injected into tumor by intratumoral way.We evaluated the prioritized ability of our materials to delivery,release and protect drugs compared with directly injected drugs.In summary,the materials synthesized in our project can be used as a carrier for transporting proteins and genes.Its biocompatibility is very good,moreover,it does not need to add other cross-linking agents during self-assembly,this process can be completed only by intermolecular hydrogen bonding.Thus our materials have a very promising outlook.On this basis,we can design a series of related structures to optimize some deficiencies of the materials,such as reduce the size of the particles,allow the materials carrying drugs into cells and releasing drugs inside cells to find other meaningful uses.