Epigenetics-inspired Photosensitizer Modification For Plasma Membrane-Targeted Photodynamic Tumor Therapy

Cancer is still the number one killer of human health,and it is still urgently needed to develop nano-drugs and methods of highly effective targeted tumor therapy to achieve the precise treatment of tumors.Although photodynamic therapy（PDT）is a new noninvasive tumor therapy,it has poor tumor selectivity and short half-life of reactive oxygen species（ROS）^[1-2].Definitely,enhancing the spatiotemporal interaction between photosensitizer and its designated target will dramatically improve the therapeutic effect of PDT.Cell membrane,the most critical protective barrier in living cells,can be regarded as a therapeutic target,which provides a new direction for tumor inhibition.The oxidation of unsaturated lipid by ROS could trigger the conformation changes of lipid membranes and cause cell death^[3-4].Compared with other organelles target PDT,plasma membrane targeting PDT was a robust strategy that directly destroyed cell integrity through photosensitive lipid oxidation without endocytosis^[5-7].However,this contact-dependent reaction between photosensitizer and plasma membrane needs to fully compromise membrane functions,and it remains a huge challenge to direct the photosensitizer to the plasma membrane of the cell through simple but universal strategies^[8-9].The amino acid methylation,acetylation or phosphorylation of histones in epigenetics prompted the chromosomal structural variation to initiate a domino effect,leading to the occurrence of epigenetic diseases^[10-13].In other words,changes in the functional modification of amino acids in histones would cause changes in the non-covalent interaction of histone and DNA complexes,thereby regulating related biological effects.Inspired by this,we speculate that a simple amino acid modification strategy may promote to regulate the subcellular distribution of the photosensitizer by enhancing the non-covalent interaction between the photosensitizer and cell membrane.ObjectiveTo explore a simple and versatile amino acid modification strategy to regulate the subcellular distribution of photosensitizers for plasma membrane targeted photodynamic tumor therapy.Methods1.A single arginine or glutamic acid modified photosensitizer was synthesized by peptide solid phase synthesis and characterized its molecular weight by ESI-MS;The plasma membrane anchoring performance of both were observed by confocal laser scanning microscope（CLSM）and some cytotoxicity tests,such as MTT method,trypan blue staining,flow cytometry and live/dead cell staining,were measured to verify the effects of their plasma membrane targeted photodynamic therapy.2.Photosensitizer conjugates modified by different amounts and types of amino acids were also composited by peptide solid phase synthesis method,which could be observed their anchoring capability under different culture time and concentration and the plasma membrane targeting effect on different cells through CLSM.Simultaneously,the mechanism of their plasma membrane anchoring was further explored.3.Photosensitizer regulated by amino acids could self-assemble into nanoparticles like NPs-E₄ and NPs-R₄,which were characterized their size distribution,zata potential and morphology by dynamic light scattering and transmission electron microscope,respectively.Moreover,Using DCFH-DA as a probe,their ROS production ability was indirectly investigate through CLSM.In addition,MTT method,trypan blue staining,flow cytometry and live/dead cell staining were used to investigate the effect of their plasma membrane targeted photodynamic therapy.Finally,the mouse breast cancer 4T1 tumor models were established to observe their biodistribution and anti-tumor effect in vivo.Results1.Compared with glutamic acid,a single arginine could regulate the plasma membrane anchoring ability of PpIX,which was beneficial to promote the therapeutic effect of photodynamic therapy on tumors.2.As the amount of arginine increased,the targeting ability of the photosensitizer conjugates was stronger,but glutamic acid was the opposite.Moreover,the membrane anchoring ability of photosensitizers regulated by amino acids depended on time and concentration and had the universality of plasma membrane targeting to different cells.Besides,this regulatory mechanism was attributed to the hydrophobic forces of PpIX and the synergistic effects of the electrostatic interactions between arginine and plasma membrane.3.The four arginine or glutamic acid could be utilized to adjust the photosensitizer to self-assemble into nanoparticles.Compared with NPs-E₄,NPs-R₄ had good stability and could improve the ability of ROS generation in cells.Benefiting from its effective ability to anchor the plasma membrane,NPs-R₄ generated ROS in situ on the plasma membrane under light irradiation,destroying the cell integrity and achieving the effect of enhancing PDT.In addition,in vivo studies confirmed that NPs-R₄ and NPs-E₄ could achieve significant tumor accumulation and effective tumor suppression with minimal systemic toxicity,but the former was more effective.ConclusionA simple and versatile amino acids modification strategy inspired by epigenetics could regulate the plasma membrane targeting of photosensitizer for enhanced photodynamic tumor therapy,which would provide new ideas for the development of structure-based drug design for site-specific delivery and tumor precision treatment.