Chimeric Peptide Engineered Exosome For Subcellular Organelle Targeted Photodynamic Therapy

BackgroundCancer is one of the leading causes of death worldwide.In recent years,over 10million new cancer cases are diagnosed every year,and around 9.6 million patients died from the disease in 2018.And according to the report of WHO,about 13 million people worldwide will die each year from cancer in 2030.Currently,different types of clinical treatments for cancer are being adopted,including surgery,radiotherapy and chemotherapy,which often fail to achieve the desired effects.Photodynamic therapy?PDT?has attracted wide attention in the application of precise tumor treatment due to its non-invasiveness and controllability.Essentially,PDT involves the administration of a tumor-localizing photosensitizer?PS?followed by local illumination of the tumor with light of a specific wavelength to activate the PS.The excited PS then transfers its energy to molecular oxygen,thus generating cytotoxic reactive oxygen species?ROS?,such as singlet oxygen?¹O₂?and free radical(O₂^�-?H₂O₂?OH^�)that can oxidize key cellular macromolecules leading to tumor cell ablation.However,?1?the photosensitizer has disadvantages such as poor water solubility and poor targeting ability;?2?Reactive oxygen species has a short lifetime??40 ns?and a limited diffusion distance??20 nm?.With the development of nanotechnology,the solubility and tumor targeting ability of photosensitizers,can be significantly improved.In recent years,targeted drug delivery systems have increasingly tended to deliver drugs into organelles to achieve more effective tumor treatment.Therefore,the role of reactive oxygen species in the treatment site will greatly improve the therapeutic effect of photodynamic therapy.The preparation process of artificial nano-carriers is complicated,and the synthesized nano-carriers often possess immunogenicity and poor biocompatibility.Therefore,targeted delivery of the photosensitizer and drug to its therapeutically active site with low immunogenicity and system toxicity is critical for optimal tumor therapy.Natural nano-sized exosomes that secreted by cells have shown great potential in drug delivery due to its good biocompatibility and low immunogenicity.Therefore,endogenous exosomes were selected as the delivery platform in this study.AimFor the sake of cell membrane and nuclear targeting function,multi-functional chimeric peptide?Chip?was synthesized and utilized to modify the surface of exosomes,to realized the nanocrystallization of photosensitizer,so as to further investigate its application in precise photodynamic therapy of tumor.MethodsSolid-phase synthesis method was used to synthesize Chimeric peptide.Exosomes,isolated and purified from the blood of SD rats by polymer precipitation technique,were modified by alkyl chain of ChiP to construct the exosome-based nano-drug delivery system?Chip-Exo?.The modified exosomes were characterized by particle size,electric potential,western blot,fluorescence intensity and the generation of reactive oxygen;in vitro experiments,we incubated Chip-Exo with tumor cells?mouse breast cancer cells,4T1 cells?and observed cellular uptake.through one stage of light,the phenomenon of photochemical internalization and lysosome escape was triggered.Finally,a two-stage light strategy was used to study the cooperative photodynamic therapy effect of Chip-Exo's double-targeted organelles.By constructing a 4T1 tumor-bearing mouse model,a two-stage illumination strategy was used to study the tumor suppressive effect in vitro.ResultsIn order to solve the defects such as poor targeting and poor water solubility of photosensitizers,standard solid-phase synthesis method was used to synthesize Chip.the ChiP of C16-K?PpIX?-PKKKRKV consists of an alkyl chain for exosome engineering,a photosensitizer PpIX for photodynamic therapy and a nuclear localization signal peptide?PKKKRKV?for nuclear translocation,which gives the photosensitizer the ability to target the cell membrane and nucleus.Confocal microscope analysis showed that the red fluorescence of Chip overlapped with the green fluorescence to cell membrane stained by DIO,indicating that Chip can effectively bind to cell membrane and has good anchoring ability of cell membrane.Exosomes were isolated and purified from the blood of SD rats by polymer precipitation technique,and then modified by Chip to construct the exosome-based nano-drug delivery system?Chip-Exo?,and the nano-photosensitizers were obtained.The modified exosomes still maintained a good size with an average diameter of 132.6 nm.The strong fluorescence signal of the modified exosomes was detected by flow cytometry,which confirmed that Chip successfully modified the surface of the exosome.In addition,the targeting ability of Chip-Exo was observed by confocal microscope.Based on the effects of membrane anchoring and lysosome entrapment,the transport of Chip-Exo in the cytoplasm and nucleus was restricted.After incubating 4T1cells with Chip-Exo for 1 h,the strategy of the first stage of light(time:30 s;intensity:29.8 m W/cm²)was adopted to trigger photochemical internalization?PCI?and lysosome escape,which significantly improved the cytoplasmic transmission of Chip-Exo.At the same time,due to the existence of nuclear localization signals?NLS?,its transmission to the nucleus is greatly enhanced.Then,the therapeutic effect of dual-stage light was evaluated by measuring the cell survival rate.After the first stage of light?1 h after incubation,time:30 s;intensity 29.8 mW/cm2?,the results showed that under the second stage of light?4 h after incubation,time:90 s;intensity:29.8 mW/cm²?,PpIX in the nucleus was activated in situ and produce reactive oxygen species?ROS?to destroy the nucleus of tumor cells,thus enhancing the therapeutic effect of PDT.Small animal imager was used to monitor the biological distribution of PpIX in real time?the dose of PpIX was 3 mg/kg/mouse?.Finally,the strategy of dual-stage light?time of irradiation:2.5+2.5 min;intensity:320 m W/cm²?was used to study the anti-tumor effect of Chip-Exo on 4T1 tumor-bearing mice.The results showed that the application of dual-stage light strategy enhanced the therapeutic effect of Chip-Exo on tumor growth.ConclusionsIn summary,multi-functional drug delivery platform?Chip-Exo?based on exosomes was successfully established,which achieved the nanocrystalization of photosensitizers and endowed them with the targeting function on cell membrane and nucleus.Both in vitro and in vivo investigations confirmed that this dual-stage light with subcellular dual-targeting strategy of ChiP-Exo can not only reduce the systemic toxicity of photosensitizers,but also significantly inhibit tumor growth.Therefore,this study provides a new strategy for the integration of diagnosis and treatment of nano-photosensitizer through the establishment of chimeric peptide-modified exosomes.