Preparation And Cytotoxicity Study Of I-131 Labling Targeted Porphyrin-liposomes

bjective:PDT is a binary cancer therapy which requires the activation of sensitizer in tissue with light locally.Porphyrin as the pioneer of photosensitizer possess a number of key photochemical,photophysical and biological properties that make them highly desirable for PDT and allows the combination of diagnosis and therapy.Nevertheless,the main challenges faced to porphyrins,including overall lipophilicity,aggregation characteristics,poor selectivity to tumorous tissue,limit their biomedical applications.To overcome those problems,various nanoparticles were development for porphyrins delivery.Loading porphyrins into nanoparticles can make hydrophobic porphyrins soluble in water and passively uptaked by cells.In addition,by conjugating specific targeting moieties or polymers to the porphyrin-based nano-carriers surface,selective accumulation can be further achieved.The most intensively investigated nano-carriers for porphyrin delivery are the bilayer structural liposomes compose of phospholipids.Those liposomes exhibit an enhanced cellular uptake due to their cellular mimic composition and structure.However,liposomes are non-specific towards tumors and their half-life are short.Thus,in this study,porphyrins were loaded to liposomes,and hyaluronic acid（HA）was selected as the cancer cell specific targeting ligand for liposome surface modification.Hence,not only increase porphyrin dispersity,but also enhance its targeting delivery.And the toxicity and targeting effect of HA-coated liposome-porphyrin（HA-lip-p）will be studied in vivo.Besides,it will be radiolabeled with I-131 to investigate the stability in vitro.We hope the radiolabeled HA-lip-p provide potential to combine PDT with SPECT and radionuclide therapy.Method:1.The hydrazide-liposome were prepared by using hydration-film method,four types of porphyrins derivatives were loaded to the liposomes respectively.The porphyrin loading efficiency was calculated according to the corresponding porphyrin standard curves measured on UV/Vis spectrometers.Dynamic light scattering（DLS）and transmittance electron microscope（TEM）were used to analysis the structure,morphology and diameter of liposome-porphyrins.The stabilities of the liposome-porphyrins were characterized by DLS after 15 mins light exposure.2.Coating of liposome-porphyrins with HA-al polymer via“click”hydrazone reaction.The cellular uptake of HA-al coated liposomes-porphyrins and free liposomes-porphyrins in MDA-MB-231 cells was observed on confocal microscopy.3.A375 and MCF-10A cells were treated with various concentration of liposomes-porphyrins,free porphyrins and free liposome under dark or light condition,cell viability was calculated by MTS assay.Meanwhile,the cytotoxicity of liposome-porphyrin to MDA-MB-231 cells was compared with or without HA-al coating under dark or light condition.4.The ¹³¹I was radiolabeled with HA-al coated liposome-porphyrins by Iodogen method,then purified with Sephadex G25 column.The radiation activity was estimated byγcounter,the radiochemical purity was determined by radioactive paper chromatography,and the vitro stability was measured in10%FBS and PBS respectively.5.Established subcutaneous model of prostate cancer in nude mice,then the ¹³¹I labeled HA-al-liposome-porphyrin and Na¹³¹I was injected into the tumor respectively,and the retention effect was evaluated by SPECT imaging.Results:1.Four kinds of porphyrins were successfully loaded in liposomes.The p-OH and p-NH₂ could be loaded in liposome more than p-P and p-py.All liposome-porphyrins showed multi-layer structure and the nanoparticle sizes of them were similar,their average particle size is 140±20 nm.The morphology changed dramatically for liposome-porphyrins after light exposure.2.HA-al was effectively coated in porphyrin-liposomes via“click”raction.We obtaind the HA-al modified lip-p-OH and lip-NH₂.3.We observed cellular uptake of lip-p-OH and lip-p-NH₂ by MDA-MB-231cells after 2 h incubation.The uptake of HA-al coated liposome-porphyrins by MDA-MB-231 cells was specific and could be block by natural HA polymer,validating that the HA-al modified liposome-porphyrins could target the CD44overexpressed cells.4.The cell viabilities were significantly reduced after incubation with Lip-p-OH and Lip-p-NH₂ for 24 h with 10 mins light exposure and higher than the free porphyrins,validating that liposome could enhance the PDT efficiency of porphyrin by increasing its dispersity.The HA coated Lip-p-OH exibited higher toxicity than the free Lip-p-OH to MDA-MB-231 cells with or without light,showing that HA coated Lip-p-OH could further enhance PDT efficiency by combining the CD44 overexpressed cells.5.The labelling rate of ¹³¹I-HA-lip-p-OH was 89%,the radiochemical purity was 94%,even the product stored in 10%FBS or PBS for 24 h,the radiochemical purity was above 80%,validating the product was stable in vitro.6.The retention effect of ¹³¹I-HA-lip-p-OH was better than Na¹³¹I.Conclusion:In this study,a HA-al modified liposome carrier was designed and synthesized for targeting delivery of ¹³¹I labeled porphyrins,in achieving a synergistic effect of SPECT imaging,radionuclide therapy and PDT.Therefore,labelling ¹³¹I with HA-al modified liposome-porphyrins appears to be a promising strategy in targeted diagnosis and treatment of tumors.