Organelles-blasting Porphyrin Photosensitizer Nanoparticles For Precise Photodynamic Therapy

Cancer is still the leading cause of death in the world today.Photodynamic therapy(PDT)is a non-invasive cancer therapy strategy in which photosensitizers generate reactive oxygen species(ROS)under tumor-localizing light activation and induce cancer cell death.However,ROS have an extremely short lifespan(0.01-45 μs)and only diffuse over a restricted distance(10-270 nm)which is much smaller than the diameter of tumor cells(10-100 μm).Thus,ROS maybe inactivated before acting on intracellular bioactive substances,contributing to the limited PDT efficiency.The above suggests that the intracellular location of ROS generation vitally affects anti-tumor efficacy.Compared with ROS generated by non-specific distributed photosensitizers,ROS generated by the organelle-precisely located photosensitizers directly act on the organelle’s bioactive substances such as protein,nucleic acid,phospholipid bilayer and,greatly shortening the diffuse distance required for function and avoiding the inactivation of ROS during the diffusion process.More importantly,direct action on the organelle’s bioactive substances of ROS damaged the organelle,causing cancer cell dysfunction,cell apoptosis,or necrosis.Mitochondrial or lysosomal-targeting PDT is the research hotspot of subcellular-targeting PDT at present.Mitochondria is the power house of tumor cells.Destroying mitochondria will cut off the energy production pathway of oxidative phosphorylation.The mitochondrial-targeting photosensitizer can produce lots of ROS at the mitochondria site,which accurately damages mitochondria and frustrate the mitochondrial oxidative phosphorylation(OXPHOS).OXPHOS inhibition reduces O2 consumption and improves ROS production of photosensitizers,greatly amplifying the OXPHOS inhibition and constructing a "closed-loop"system.However,the anticancer efficiency of OXPHOS inhibition remains unsatisfactory,mainly because cancer cells utilize OXPHOS and glycolysis to meet the energy supply demand.Even worse is that if one pathway of energy metabolism is blocked,the other would undergo a compensatory activation to rescue the energy loss.Therefore,cutting off simultaneously the two energy metabolism pathways of OXPHOS and glycolysis is expected to achieve fatal energy depletion of tumor cells.Pyropheophorbide a(PPa)was conjugated with triphenylphosphine to construct mitochondrial-targeting photosensitizer(TPPa)to explore its capacity to block OXPHOS.Syrosingopine(Sy),as a glycolysis inhibitor,combined with TPPa to induce lethal energy depletion of tumor cells.In vitro synergistic cytotoxicity,the combination of TPPa and Sy showed significant synergistic effect and the mitochondrial targeting capacity of photosensitizer was the key to the synergistic effect.To ensure the efficient accumulation of drugs at the tumor site,we constructed biomimetic PLGA nanoparticles(mTPPa-Sy NPs)in which the PLGA nanoparticles load with TPPa and Sy as the core.and the tumor cell membrane as the biomimetic surface.The mTPPa-Sy NPs showed uniform particle size and distribution.In vitro cytotoxicity and synergistic mechanism of mTPPa-Sy NPs was investigated.TPPa accurately located mitochondria and generated lots of ROS under laser irradiation,destroying the mitochondrial respiratory electron transmission chain(ETC)and blocking OXPHOS.Sy inhibited MCT-1 mediated lactate efflux,disrupting glycolysis in cancer cells.mTPPa-Sy NPs cut off two energy production pathways concurrently,which effectively causes fatal energy depletion of tumor cells.And the precise damage of mitochondria was crucial to the energy depletion of tumor cells induced by mTPPa-Sy NPs.The pharmacokinetic behavior and biodistribution of mTPPa-Sy NPs was investigated.Owning that the cloak of tumor cell membrane endowed the nanoparticles with immune escape capacity,the mTPPa-Sy NPs significantly improved the pharmacokinetic behavior of TPPa.Moreover,the mTPPa-Sy NPs showed a distinctly higher concentration than other groups at tumor sites and reached the peak concentration at 24 h.The enhanced intratumoral distribution and the extended peak time was attributed to the prolongation of systemic circulation time and homologous targeting of the tumor cell membrane.The anti-tumor efficacy of mTPPa-Sy NPs was evaluated in vivo.After a single administration,the mTPPa-Sy NPs exhibited the most potent tumor-inhibiting efficiency,with significantly reduced tumor volume.The efficient tumor inhibition was mainly attributed to two aspects:(1)The immune escape and tumor homologous targeting of mTPPa-Sy NPs benefits the efficient intratumoral accumulation of drugs.(2)mTPPa-Sy NPs could simultaneously cut off two energy production pathways of tumor cells,causing fatal energy depletion of tumor cells.Compared with those in normal cells,the lysosomes in tumor cells are more numerous,larger,more fragile,and contain a lot of cathepsins with higher activity.The release of cathepsins from the lysosome into the cytoplasm could trigger cancer cell death by apoptosis and apoptosis-like pathways.This release occurs by lysosome membrane permeabilization(LMP).Lysosome-targeting PDT could induce extensive LMP and lead to intense cancer cell cytotoxic.Therefore,we constructed four kinds of lysosome-targeting photosensitizers(LPPa)by linking PPa with tertiary alkyl amine,named PPal,PPa4,PPa8,and PPa12 respectively.PPa was selected as the model drug,and tertiary alkyl amines with different carbon chain lengths(one-carbon chain length C1,four-carbon chain length C4,eight-carbon chain length C8,and twelve-carbon chain length C12)were selected as lysosomal targeting ligands.The four designed LPPa self-assemble into uniform nanoparticles(LPPa NPs).Interestingly,with the increase of the side chain length of alkyl tertiary amine,the self-assembly ability of LPPa increased.PPa8 had the highest self-assembly ability among four designed LPPa,attributed to the double effect of hydrophobic force and electrostatic force.Lysosomal localization of LPPa NPs was investigated.Owing to the diverse interaction between different alkyl tertiary amines and the lysosomal membrane,four LPPa had different distribution in the cancer cells.Under acidic conditions of lysosome,the tertiary amine structure of the alkyl tertiary amine protonated and bound to the lysosomal membrane,benefiting the interaction of alkyl groups and the phospholipid bilayer of the lysosomal membrane.The carbon length of alkyl groups determines the distribution of LPPa at the lysosome.The short-chain alkyl groups couldn’t disturb the phospholipid bilayer of the lysosomal membrane so that the PPal and PPa4 with the short-chain alkyl tertiary amines anchored on the lysosomal membrane and displayed accurate lysosomal localization.Long alkanes could disrupt the phospholipid bilayer of the lysosome membrane,so that PPa8 and PPa12 with long-chain alkyl tertiary amines could successfully escaped during the process of bilayer permutation.The ROS generation ability and cytotoxicity of LPPa NPs was investigated.PPa4 NPs had the strongest ROS production capacity and showed the most potent cytotoxicity,attributed to the improved cellular uptake,accurate lysosomal localization and high ROS generation capacity of PPa4.Compared with PPa NPs,LPPa NPs significantly improved the AUC of the photosensitizer.The modified alkyl tertiary amine enhanced the self-assembly ability of the photosensitizer and improved the stability of nanoparticles in vivo,improving pharmacokinetic behavior.PPa8 NPs had the best self-assembly ability and displayed the highest AUC.The tissue distribution of LPPa NPs was investigated.Compared with PPa solution and PPa NPs,LPPa NPs enhanced tumor accumulation and prolonged optimal tumor accumulation time of photosensitizers.PPa8 NPs had the highest tumor accumulation,attributing to the highest assembly ability and the highest AUC of PPa8 NPs.The anti-tumor effect of LPPa NPs in vivo was investigated.Compared with PPa solution and PPa NPs,LPPa NPs had a stronger anti-tumor efficiency.PPa4 NPs displayed the most potent tumor-inhibiting efficiency,which can be attributed to three points.Firstly,PPa4 has the highest ROS generation ability owing to the closest optical properties to PPa.Secondly,PPa4 improved the AUC and intratumoral accumulation of photosensitizer attributed to enhanced assembly ability.Finally,PPa4 could precisely located lysosomes,causing lysosomal membrane permeabilization and inducing efficient tumor cell death.