The Research Of Semiconducting Polymer-based Photodynamic Therapy On Human Gastric Cancer

Background：Gastric cancer was one of the most malignant tumors, had high incidence inChina. The2013Chinese cancer registry annual report showed that the mortality rateof gastric cancer in2010accounted for the third of malignant tumors mortality. Earlygastric cancer had no symptoms or only slight symptoms, while had obvious clinicalsymptoms, the tumor had already advanced. Currently, surgical resection,endoscopic resection, radiotherapy and chemotherapy drugs were still the maintreatment. Unfortunately, some patients despite were treated by the above treatment,but were still not ideal. Photodynamic therapy (PDT) as a minimally invasivetreatment had gained more and more attention.This method used specificwavelengths of light to excite the photosensitizer accumulated in the tumor tissue,produce cytotoxic factors, resulting in tumor cell damage and death, in order to curecancer. Thus, photosensitizers were the key factor for PDT.The organic semiconductor polymers (also known as polymer dots) were due toπ-conjugated electrons having semiconductor properties, such as very large opticalabsorption cross section, high fluorescence quantum efficiency, which exciting byappropriate wavelength light could transfer energy from polymers to photosensitizeror other materials generating singlet oxygen through fluorescence resonance energytransfer. The polymers had flexible hydrophobic characteristics inside could solveproblems in many water-soluble drugs, while external hydrophilic segment had astabilizing effect in water. In addition, the fluorescence properties of thesemiconductor polymers could help track the location of the photosensitizer, toachieve localization of the photosensitizer and real-time monitoring. These featuresmade these miconductor polymers as carrier for photosensitizer compareing with the traditional nanocarriers needed to release the loaded photosensitizer had obviousadvantages.Objectives：In this study, we use semiconducting polymer as the carrier of photosensitizerby nanoprecipitation encapsulating hydrophobic photosensitizer into the polymer,then prepare photosensitizer-doped polymer nanoparticles (referred as TPP-dopedPFBT nanoparticles). In vitro and in vivo experiments, research the distribution ofTPP-doped PFBT nanoparticles in human gastric cancer cells and tumor-burdenedmice, and its mediated photodynamic effects on tumor cells and tumor tissues,discuss the feasibility of nanoparticles mediated PDT on treatment of stomachcancer.Methods：⑴Using nanoprecipitation method to prepare TPP-doped PFBT nanoparticles.Using UV-Vis scanning spectrophotometer to measure absorption value ofnanoparticles, and then calculate the concentration of nanoparticles solutionaccording to absorption values. The nanoparticle size and size distribution weremeasured by the dynamic light scattering. The morphology of nanoparticles wasmeasured by the transmission electron microscopy. UV-Vis absorption spectra andfluorescence spectra were measured by UV-Vis scanning spectrophotometer andfluorescence spectrometer, respectively. Under50mW/cm2blue LED light, singletoxygen production of nanoparticles’ solution was detected by ADMA.⑵In vitro experiment, the localization and uptake in tumor cells could beobserved/measured by fluorescence microscope and flow cytometry, according tothefluorescence properties of nanoparticles themselves. The dark toxicity and killingeffects of the TPP-doped PFBT nanoparticles on human gastric adenocarcinomaSGC-7901cells were measured by MTT assay, while observing the time ofnanoparticles interact with cells, nanoparticles’ concentration and the light doseimpact on the effectiveness of PDT. The cell morphology, intracellular reactiveoxygen species, mitochondrial membrane potential and nuclear morphology afterPDT were performed on optical microscope and fluorescence microscope.⑶Establish human gastric cancer xenograft model in nude mice, and observe the distribution of the TPP-doped PFBT nanoparticles in the tumor-burdened miceby vivo imaging system. Observe the inhibition and killing effects of TPP-dopedPFBT nanoparticles mediated PDT on the tumor-burdened mice and the differencesbetween them via different routes of injection. The tumor, liver, spleen, kidney, lungand heart were removed, fixed by10%marin buffer, sliced after embedding paraffin,stained by hematoxylin-eosin and observed morphological changes by opticalmicroscope after the PDT treatment.Results：⑴The TPP-doped PFBT nanoparticles were prepared successfully, hadspherical shape, about24nm in diameter,460nm absorption wavelength,625-750nmemission wavelength. When TPP-doped PFBT nanoparticles were excited by460nmblue light, could transfer energy from polymer PFBT to photosensitizer TPP throughfluorescence resonance energy transfer, emitted red fluorescence. The ADMAdetection showed the singlet oxygen production of nanoparticles’ solution waspositively correlated with the irradiation time, reach the plateau at13min.⑵In vitro experiment, we found that TPP-doped PFBT nanoparticles couldthrough the SGC-7901cell membrane, and locate in the cytoplasm. With theincrease the concentration of the nanoparticles, SGC-7901cell uptake the amount ofnanoparticles increasely, and the number of cells uptake nanoparticles also increase.MTT results showed that the TPP-doped PFBT nanoparticles interact with humantumor cells or human normal cells were no dark toxicity under the condition of nolight. The killing effects by nanoparticles mediated PDT on human gastricadenocarcinoma SGC-7901cells, was positively correlated with incubation time,nanoparticles’ concentration and light dose. The results of optical microscope andfluorescence microscope showed SGC-7901cells apoptosis and necrosis after PDT,such as cell shrinkage becomed round and small, appeared in the cytoplasm vacuoles,increased intercellular space, refraction, adherent ability decline, nucleus chromatinpyknosis, a lot of grain material and debris. Intracellular reactive oxygen speciesincreased and mitochondrial membrane potential was damaged after PDT.⑶The vivo experimental results showed that TPP-doped PFBT nanoparticlesintravenously injected in tumor-burdened mice mainly distributed liver and spleen, less in tumor tissue, while nanoparticles locally injected in the tumor site mainlyaccumulated in tumor tissue.TPP-doped PFBT nanoparticles mediated photodynamictherapy on tumor-burdened mice had obvious inhibition and killing effects, includinglarge areas of coagulation necrosis in the tumor tissues, tumor cells arranged incrumb, inflammatory cells infiltration, capillary reduced, especially local injection.TPP-doped PFBT nanoparticles intravenously injected in tumor-burdened mice hadliver damage and no damage in other organs.Conclusions：⑴The TPP-doped PFBT nanoparticles had good development prospectbecause of uniform size, small particle size, no cellular dark toxicity, and thepolymer could transfer energy for internal photosensitizer, emitted red fluorescenceand generated singlet oxygen.⑵The TPP-doped PFBT nanoparticles mediated PDT had obvious therapeuticeffects on SGC-7901cellsin vitro and tumors on tumor-burdened mice, resulting incells apoptosis and necrosis.⑶The TPP-doped PFBT nanoparticles had passive targeting property, but hadliver damage when injected intravenously, so should be further improved itspharmacokinetic properties in subsequent experiments to increase active targeting,improve therapeutic effects, and reduce side effects.