Graphene Oxide Photosensitizer Complex Based Photothermal And Two-photon Photodynamic Therapy Aginst Cancer

Cancer is one of the major threats to human health and challenges faced by modern medicine.There are three main conventional methods against cancer at present,including surgery,radiation therapy and chemotherapy.Photodynamic therapy is also increasingly applied to the treatment of cancer as a noninvasive therapy.PDT utilizes photosensitizers(PS)which can be activated by a certain wavelength light source to transfer energy to surrounding oxygen,thus generating reactive oxygen species(ROS)which can result in oxidative damage to tumor cells and finally kill them to achieve therapeutic purposes.Compared with surgical treatment,PDT is a noninvasive therapy which can reduce the pain of patients,and it rarely lead to drug resistance as chemotherapeutic drugs.However,due to the physical and chemical properties of photosensitizer,they can normally be excited by the light with wavelength 600-700 nm,penetration power of these short-wavelength light is comparatively weak that it can't reach deep-seated tumor,so the clinical application of photodynamic therapy is limited to the skin diseases or superficial tumor currently.Moreover,most of the photosensitizer is difficult to dissolve in water,and easy to accumulate in the body,affecting the tumor targeting and the generation of singlet oxygen in vivo,thereby decreasing the photodynamic therapy efficiency for cancer treatment.At present,the emerging two-photon photodynamic therapy can use two-photon absorption(2PA)compounds to absorb energy from near-infrared light source and then transfer energy to photosensitizer through fluorescence resonance energy transfer(FRET)effect to produce reactive oxygen species such as singlet oxygen.Therefore two-photon photodynamic therapy can overcome the shortcomings of photodynamic therapy with poor penetration in theory.Graphene oxide(GO),the derivative of graphene,has been widely investigated in the field of biological medicine,such as drug delivery,disease diagnosis,tissue engineering and gene transfection because of its unique optical properties and thermal properties.It has a huge surface area with monolayer two-dimensional structure which is able to load aromatic compounds by ?-? stacking,such as hydrophobic photosensitizers with conjugated aromatic ring,which can improve their water solubility.Due to a lot of hydroxyl,carboxyl,epoxy groups and other oxygen-containing groups on the surface of GO,it can be functionally modified,thus making it even wide application.In this paper,pegylated graphene oxide(GO-PEG)was used as drug delivery carrier to co-load the photosensitizer pyropheophorbide a(PPa)and pyridine 2PA compound BL4(GO-PEG-PPa/BL4).BL4 and PPa can be excited by near-infrared laser(980 nm)through FRET effect after releasing from the drug delivery system.Meanwhile,GO can play the role of photothermal therapy by photothermal conversion under the same laser's excitation.Here,the therapeutic effect of GO-PEG-PPa/BL4 combined photothermal therapy with two-photon photodynamic therapy against cancer in vitro and in vivo was evaluated on 4T1 mouse breast carcinoma cells and mouse subcutaneous tumor model respectively.The relevant research work of this paper was carried out from the following three parts.In the first part,a drug delivery system based on graphene oxide was constructed.Firstly,some carboxyl groups on the surface of GO were covalently bonded with PEG in order to obtain GO-PEG.The products was characterized by atomic force microscopy,ultraviolet-visible spectrophotometry,infrared spectrophotometry and zeta potential.GO-PEG loading with PPa and BL4(GO-PEG-PPa/BL4)was prepared by non-covalent interaction such as ?-? stacking and hydrophobic effect.The encapsulation efficiencies of PPa and BL4 were determined by ultraviolet-visible spectrophotometry and fluorescence spectrophotometry.And the generation of singlet oxygen in vitro before and after drug-loaded was detected by reagent anthracene-9,10-dipropionic acid disodium salt(ADPA).Then the in vitro release behavior of GO-PEG-PPa/BL4 in different pH buffers and temperature-rising effect of GO samples under near infrared laser irradiation(980 nm)were investigated successively.The results showed that GO-PEG could co-load with PPa and BL4,and the encapsulation efficiencies of PPa and BL4 were 36 % and 30 %,respectively.PPa and BL4 could produce singlet oxygen in solution instead of loading onto GO-PEG.PPa and BL4 could be released simultaneously at different pH values.Under irradiation of near-infrared laser,GO can absorb the light energy and convert it into heat energy,that is,it has certain photothermal conversion capability.In the second part,the anti-cancer activity of the drug delivery system was studied.The light/dark cytotoxicity of GO-PEG-PPa/BL4 was investigated on 4T1 mouse breast carcinoma cells.The intensity and time of lighting were optimized for the phototoxicity experiments.The efficacy of photothermal and two-photon photodynamic therapy was evaluated via MTT assays and the apoptosis population of cancer cells was measured by flow cytometry.Finally,the cellular uptake of GO-PEG-PPa/BL4 was investigated.The results showed that GO,GO-PEG and BL4 had no significant toxicity to cells,and the dark cytotoxicity of photosensitizer PPa decreased after loaded by GO-PEG.GO-PEG-PPa/BL4 could perform the combination of photodynamic therapy and two-photon photodynamic therapy on cells.GO-PEG-PPa/BL4 can be uptaken by cells and had a certain time-dependent manner.In the third part,the subcutaneous model of mouse breast carcinoma was established and the efficacy of the drug delivery system was investigated.The tumor models in Balb/c mice was established by subcutaneously injecting 4T1 cells.The distribution of GO-PEG in vivo was investigated,and then the antitumor effect of photothermal and two-photon photodynamic therapy was studied on tumor mice.After that,the main organs of mice such as heart,liver,spleen,lung and kidney were sectioned to H&E staining test.In addition,H&E staining and TUNEL staining were performed on mouse tumor tissue sections.The experimental results indicated that GO-PEG-PPa/BL4 laser group could effectively inhibit the growth of tumor in mice,followed by GO-PEG laser group.There was no significant damage to the main organs of mice in experimental group and control groups,which indicated that the photothermal and two-photon photodynamic therapy based on graphene oxide had good biocompatibility.The results of TUNEL staining also showed that GO-PEG-PPa/BL4 had the best antitumor effect,which explained the macroscopic results of photothermal and two-photon photodynamic therapy against cancer.In summary,the study preliminarily validated that the GO-PEG-PPa/BL4 delivery system had a good photothermal and two-photon photodynamic therapy effect and certain safety through a series of experiments both in vitro and in vivo.This work sheds light on a new means for cancer treatment in the future.