Construction And Evaluation Of A Self-sensitized Photodynamic Therapy System Based On Bioluminescence Resonance Energy Transfer

BackgroundCancer is one of the main diseases threatening human life and health.In order to overcome the shortcomings of mainstream cancer therapies such as surgery,chemotherapy and radiotherapy,new treatment strategies are urgently needed.Photodynamic therapy(PDT)is considered to be a promising treatment because of its advantages of less invasion,less drug resistance and less side effects.The mechanism of PDT is that reactive oxygen species(ROS)produced by PS activated by external light can kill cancer cells.Photosensitizer(PS),light source and oxygen(O₂)are the three basic elements of PDT.However,in clinical practice,PDT can be used only for the treatment of superficial cancer in most cases.The main reasons are that the excitation wavelength of many PSs locate in the visible region,and the exogenous light source in the region has a shallow penetration depth;meanwhile,if cancer locates inside the body,the effect of PDT will be weakened or invalidated owing to the limited accessibility and accurately of light sources.Some studies have proposed strategies to construct light sources in cancer tissues,such as introducing chemiluminescence materials to make cancer cells produce endogenous light,or introducing luciferase and its substrates to induce cancer cells to produce bioluminescence.Both of them deliver exogenous chemiluminescence materials or luciferase into cells.PurposeWith the development of gene delivery technology and nano drug delivery technology,we proposed to transfer luciferase gene into cancer cells to express luciferase,and constructed a PDT system based on bioluminescence resonance energy transfer(BRET-PDT).Different from traditional PDT,the system can activate photosensitizer without external light source.Method1.To construct BRET-PDT system,firefly luciferase gene plasmid(FLase-GP)was transfected into cancer cells to express firefly luciferase(FLase),and the substrate of firefly luciferase,fireflyluciferin(FLuc),was transfected into cancer cells,FLuc is introduced into cancer cells to produce bioluminescence and activate hypericin(Hyp,as PS)to produce ROS.As a predictable result,the innovative PDT system would realize the cytotoxicity on cancer cell and solid cancer tissue without exogenous light source.The feasibility of the system was verified by gene transduction efficiency,target protein FLase production,photon production and cytotoxicity in vitro.2.Using nano-drug delivery technology,the photosensitizer Hyp and FLase substrates FLuc were prepared using carboxymethyl chitosan(CMC)and polylactic-glycolic acid copolymer(PLGA)as carriers respectively to prepare nanoparticles:Hyp-CPNPs And FLuc-CPNPs.And carried out physical and chemical characterization,including nanometer morphology and particle size,Zeta potential,infrared absorption and X-ray diffraction and other indicators,and pharmaceutical characteristics research,including drug loading rate and encapsulation rate test,in vitro drug release test.3.The in vitro cytotoxicity of drug delivery materials,drug loaded nanoparticles and free drugs are compared.4.The cell uptake,subcellular localization,ROS generation and apoptosis experiment are performed to explore the mechanism of the cytotoxicity produced by the innovative BRET-PDT system.Result1.Firstly,it is proved that FLase-GP can efficiently express the target protein FLase after it introduced into He La cells.It is also confirmed that FLase expressed in the cell can act on its substrate FLuc to produce photons.The generated photons activate Hyp to produce ROS.Subsequently,ROS produces cytotoxicity by causing apoptosis or necrosis of cancer cells.2.We successfully prepared nanoparticles to load FLuc and Hyp,named FLuc-CPNPs and Hyp-CPNPs,respectively.Under the scanning electron microscope,they are spherical,with smooth surface and uniform distribution,with particle sizes of 173.30±0.38 nm and190.20±2.63 nm.The Zeta potentials are-18.70±6.97 m V and-8.66±4.44 m V,respectively.The infrared spectroscopy and X-ray diffraction results show that the two compounds are well loaded in the nanoparticles.For Hyp-CPNPs,the drug loading efficiency and encapsulation efficiency reached 22.60%and 72.76%,respectively.For FLuc-CPNPs,the drug loading efficiency and encapsulation efficiency reached 16.64%and52.16%,respectively.Under the p H condition of the tumor microenviro nment(p H=6.5),the release of Hyp within 24 h was 69.4%,and at 48 h,the cumulative release finally reached78.1%.3.The results of biological effects show that the drug delivery materials have no cytotoxicity,and Hyp-CPNPs do not produce cytotoxicity without light source activation when Hyp-CPNPs are activated by an external light source,or activated by an internal light source in the presence of FLase-GP and FLuc-CPNPs,obvious cytotoxicity were observed.4.Both FLuc-CPNPs and Hyp-CPNPs enter cells quickly,and Hyp-CPNPs are enriched in mitochondria in cells.In the presence of FLase-GP and FLuc-CPNPs,Hyp is activated by the endogenous light source to generate ROS and cause cell apoptosis.ConclusionWe successfully constructed and verified the integrity of the new self-luminous BRET-PDT.The prepared FLuc-CPNPs and Hyp-CPNPs can be used for excellent drug delivery system.BRET-PDT has excellent in vitro cancer cell toxicity,Hyp-CPNPs can quickly enter cells and localize in mitochondria.In the presence of FLase-GP and FLuc-CPNPs,it can produce ROS and cause cell apoptosis.These results indicate that the internal light mediated BRET-PDT is feasible.This may become a new strategy of PDT.