Molecular FRET Co-Assembly Of Biomimetic Nanoparticles For Synergistic Phototherapy Of Triple-Negative Breast Cancer

Triple Negative Breast Cancer(TNBC)is poorly sensitive to chemotherapy due to the lack of key receptors,resulting in poor clinical efficacy.Photothermal therapy(PTT)and photodynamic therapy(PDT)induce tumor cell apoptosis and necrosis leveraging local temperature rise or generating reactive oxygen species(ROS).As they do not depend on the expression of cell-specific receptors,they could be a good choice for TNBC treatment.However,the antitumor effect of PTT alone is limited,and PDT is limited by local hypoxia in the tumor,resulting in inefficient production of reactive oxygen species.Combined PTT with PDT can not only induce local temperature rise to kill tumor cells,but also improve local blood circulation,relieve tumor hypoxia,and promote the production of ROS.In addition,patients need to be protected from light after using PDT photosensitizers to prevent the toxicity of ROS generated by light to normal tissues.Therefore,how to realize the efficient combination of PTT and PDT and minimize the phototoxicity of photosensitizers will realize the "high efficiency and low toxicity" combination therapy for TNBC.In response to these problems,this research leverage molecular assembly nanotechnology,fluorescence resonance energy transfer(FRET)between specific photosensitizer molecules and biomimetic technology of red blood cell membrane coating,to construct a PTT@PDT molecular assembly biomimetic nano-drug delivery system.The typical organic photodynamic/photothermal photosensitizer were selected to validate the FRET-based molecular assembly concept,and Ce6@DiR co-assembled nanoparticles were prepared using Ce6(FRET donor)/DiR(FRET acceptor)as model photosensitizers,and erythrocyte membrane biomimetic coating(Ce6@DiR-M NPs)was performed on the coassembled nanoparticles.The in vitro photothermal effect of Ce6@DiR-M NPs and the effect of this process on the photodynamic activity of Ce6 were investigated,as well as their in vitro antitumor activity.The in vivo pharmacokinetic behavior and tissue distribution properties of Ce6@DiR-M NPs were investigated,as well as their in vivo photothermal effects and regulation of tumor hypoxia.Special focus was given on the therapeutic effect of Ce6@DiR-M NPs in vivo programmed synergistic phototherapy in the mouse 4T1 tumor model,and the effect of Ce6@DiR-M NPs on the phototoxicity of normal tissues.First of all,based on molecular assembly nanotechnology,four representative organic photodynamic photosensitizers(PDP)and photothermal photosensitizers(DiR)were selected to prepare co-assembled nanoparticles by a one-step nanoprecipitation method.Driven by supramolecular forces such as hydrophobic interaction and π-π stacking,all these PDP formed uniform nanostructures with DiR,indicating that this molecular assembly technology has great potential for efficient delivery of hydrophobic organic photosensitizers.In addition,as DiR can completely cover the emission band of these PDP,DiR can be the FRET acceptor for these PDPs.The FRET interaction between DiR and PDPs significantly reduces the ROS generation of PDPs,indicating that this FRET-based molecular assembly technology is a solution to alleviate the potential normal tissue phototoxicity of PDP.Ce6@DiR co-assembled nanoparticles were prepared using chlorin e6(Ce6)as a model photodynamic photosensitizer,and the co-assembled nanoparticles were biomimetic coated with erythrocyte membrane,and the particle size of the co-assembled nanoparticles before and after coating was investigated.Zeta potential and formulation stability,and the effect of formulation process on membrane-related proteins were investigated.After screening,the molar ratio of Ce6 to DiR was determined to be 1:1.5.The average diameter was about 97.5 nm,and the Zeta potential was about -10 mV.Red blood cell membrane biomimetic coassembled nanoparticles(Ce6@DiR-M NPs)were prepared by extruding a mixture of Ce6@DiR co-assembled nanoparticles and red blood cell membrane(RBCM)at a mass ratio of 1:5.Transmission electron microscopy images revealed the successful preparation of Ce6@DiR-M NPs,and western blotting revealed the presence of typical CD47 molecules on the surface of Ce6@DiR-M NPs.Furthermore,Ce6@DiR-M NPs exhibited good colloidal stability in PBS containing 10%FBS.The in vitro photothermal effect of Ce6@DiR-M NPs and the effect of this process on the photodynamic activity of Ce6 were investigated.The temperature of the Ce6@DiR-M NPs solution can easily reach above 40-50℃ after 808 nm laser treatment.And during this process,the nanostructure of Ce6@DiR-M NPs is destroyed.Under cascaded laser irradiation(808 nm→660 nm),the Ce6@DiR-M NPs solution achieved a singlet oxygen yield comparable to that of the Ce6 solution.These results confirmed that after Ce6@DiR-M NPs exerted the photothermal effect,DiR was depleted by photobleaching,and the quenched Ce6 in the assembly could effectively restore the photodynamic activity.The cellular uptake and in vitro killing activity of Ce6@DiR-M NPs against mouse triplenegative breast cancer cells 4T1 were investigated.Only very weak Ce6 fluorescence signal was observed after 2 hours of incubation between Ce6@DiR-M NPs and 4T1 cells,but when the cells were treated with 808 nm laser(1 W/cm~2,5 min),the fluorescence intensity was significantly recovery,indicating that the nanoparticles have good uptake and that the quenched Ce6 in the nanoparticles can restore the photoactivity after photobleaching of DiR.In addition,Ce6@DiR-M NPs exhibited negligible cytotoxicity under 660 nm laser irradiation(1.5 m W/cm~2,5 min)compared with Ce6 solution.The cytotoxicity of Ce6@DiR-M NPs was significantly increased after photobleaching of DiR by 808 nm laser(1 W/cm~2,5 min).Furthermore,combined treatment of PTT and PDT using 808 nm laser and 660 nm laser significantly enhanced killing of 4T1 cells compared to single phototherapy.The effect of photothermal on the penetration of photosensitizers was investigated using a tumor cell spheroid model.After incubation of tumor spheroids with Ce6 solution and Ce6@DiR-M NPs as well as 808 nm laser treatment,the Ce6@DiR-M NPs incubation group showed stronger fluorescence in the deep part of tumor spheroids.Increased temperature and partial apoptosis resulted in increased space between cells in the spheroid.These results confirmed that DiRinduced increase in tumor site temperature could not only enhance the antitumor effect,but also promote the penetration of photosensitizers into tumor tissue.Using SD rats and 4T1 tumor-bearing mice as models,the pharmacokinetic behavior and tissue distribution characteristics of Ce6@DiR-M NPs were investigated.After intravenous administration,the DiR solution were rapidly cleared from the body.In contrast,Ce6@DiR nanoparticles could prolong the circulation time in the blood.Compared with DiR solution and Ce6@DiR NPs,erythrocyte camouflaged nanoassemblies(Ce6@DiR-M NPs)significantly prolonged the retention time of DiR in blood,even better than PEGylated nanoparticles(Ce6@DiR-PEG NPs).More importantly,Ce6@DiR-MNPs can effectively solve the dilemma of PEGylation modification.The clearance rate of the second dose of Ce6@DiR-PEG NPs was significantly accelerated,while the second dose of Ce6@DiR-M NPs did not change much.The improved pharmacokinetic behavior of Ce6@DiR-M NPs can be attributed to the improved stability and immune escape of RBCM.CD47 expressed on RBCM is a "don’t eat me" marker that allows Ce6@DiR-M NPs to escape from immune clearance.The biodistribution was explored in 4T1 tumor-bearing mouse xenograft model.Negligible fluorescent signal was found in tumors of DiR solution-treated mice.In contrast,both Ce6@DiR NPs and Ce6@DiR-M NPs showed a significant increased fluorescence,and Ce6@DiR-M NPs showed higher fluorescence signals than Ce6@DiR NPs in tumor accumulation.The increased intratumoral accumulation of Ce6@DiR-M NPs should be attributed to the longer systemic circulation time and enhanced permeability and retention effect.In vivo photothermal effect and regulation of tumor hypoxia of Ce6@DiR-M NPs were then investigated.After 808 nm laser irradiation at the tumor site post administration,Ce6@DiR-M NPs exhibited a more pronounced temperature increase at the tumor site compared to Ce6@DiRNPs and DiR solution due to better tumor accumulation.Elevated tumor local temperature has been found to help alleviate tumor hypoxia and improve PDT efficiency.Therefore,hypoxia probes were used to further evaluate tumor hypoxia status after photothermal therapy.The results showed that 4T1 tumor-bearing mice without laser irradiation presented large areas of tumor hypoxia in tumor tissue.In contrast,tumor hypoxia was significantly relieved after administration and 808 nm laser irradiation(2 W/cm~2,5 min),and the relieved hypoxia was beneficial to the PDT efficacy of Ce6.The effect of Ce6@DiR-M NPs under programmed synergistic phototherapy in the mouse 4T1 tumor model was investigated as well as protection effect against the phototoxicity for normal tissues.The tumor site was treated with 808 nm laser irradiation 12 hours post injection to promote photosensitizer deep penetration and relieve tumor hypoxia,and further treated programmed laser irradiation(808 nm→660 nm)24 hours post injection for combined treatment of PTT and PDT.Under this treatment strategy,Ce6@DiR-M NPs-treated mice showed almost no significant progress in tumor growth,while the monotherapy-treated group showed only a weak therapeutic effect,demonstrating the synergistic effect of this biomimetic nanoassembly delivery technology.In addition,during the treatment,no obvious abnormal function of liver and kidney or tissue damage were found.We further investigated whether Ce6@DiR-M NPs could control the phototoxicity of ROS to normal tissues.Depilated healthy Balb/c mice were exposed to 660 nm laser for 2 h after injection of Ce6 solution and Ce6@DiRM NPs.It was observed that compared with the Ce6@DiR-M NPs-treated group,the skin and kidneys of Ce6 solution-treated mice had obvious necrotic areas,indicating that Ce6@DiR-M NPs could effectively prevent the phototoxicity of ROS to skin and other superficial tissues during PDT treatment.