Synthesis And Activity Study Of Chlorophyll A Based Photosensitizers

Photodynamic therapy(PDT)stimulates photosensitizers(PSs)enriched in target tissues by light of a specific wavelength to produce cytotoxic reactive oxygen species(ROS)to kill target cells.In the clinical treatment of malignant tumors,PDT has developed into a routine modality,but it also faces challenges such as limited treatment depth,unclear structure-activity relationship,and unsatisfactory tumor targeting.Based on internal Spirulina resource,this paper focused on developing novel long wavelength chlorophyll a derived PSs,and also tumor acidic microenvironment responded dual chlorin dimer,aiming at improving the tissue penetration and efficacy of PDT,as well as realizing simultaneous fluorescence imaging and PDT treatment.Meanwhile,our study on photoreaction mechanism,cellular uptake,organelle localization and PDT activity of modified PSs offered reference to understanding the structure-activity relationship of this kind of PSs.By optimizing the previous preparation process of CHC(Chenghai-Chlorin)from Spirulina platensis produced in Chenghai Lake of Yunnan Province,the purity of crude product reached to 91%,consistent with domestic commercial products.Besides,CHC copper complex CHCTME-Cu was prepared and the C-17 and 18 were both confirmed to be S configuration by X-ray single crystal diffraction,the same with marine derived Chlorin e6.In addition,the composition of crude CHP was confirmed with LC-MS analysis.The treatment depth of PDT depends on the maximum absorption wavelength of the photosensitizer used.Aromatic groups were introduced at the 32 position of CHC to obtain 3-3a-f with a significant red shift of maximum absorption(7?14 nm),through improved regio and stereoselective bromination strategy and Pd-catalyzed coupling reaction.Aryl substitution increased PDT potency of the modified product by 12?58 folds compared with CHC by enhancing the drug accumulation in HepG2 cells and the level of type ? reactive oxygen species,despite reduced singlet oxygen yield.With the 32-aryl substitution retained,amino acid side chains were respectively introduced at the 131,152 and 173carboxylic acids of CHC by amide condensation to obtain 16 novel long-wavelength CHC amino acid conjugates.The conjugated aromatic substitution also resulted in a maximum absorption red shift by 6 to 10 nm.Studies on reactive oxygen species in aqueous solutions detected by fluorescent probes showed that:aryl substitution promoted the production of type ? reactive oxygen species while reducing the production of singlet oxygen;amino acids affected the photoreaction process by changing the molecule net charge,with the increase of positive charge and the total charge number respectively facilitating the type ? and type ?photoreaction;in addition,the singlet oxygen rather than the type ? reactive oxygen species level determined the rate of photodegradation of these compounds.Fluorescence colocalization studies between photosensitizers and organelle probes showed that this kind of photosensitizers were preferentially located in lysosomes(Pearson correlation coefficient 0.602?0.826),and the cation basic amino acids conjugates had slightly higher mitochondrial distribution than the anion acidic amino acid derivatives(Pearson coefficients 0.368?0.503 and 0.244?0.398,respectively).Thanks to a significantly increased cellular uptake,the in vitro PDT efficacy against HepG2 cells of modified products were 74-241 folds than CHC,among them,4-48a demonstrated low toxicity and high potency(IC50=0.23±0.01 ?M,dark/phototoxic ratio>870),different from the other derivatives.A dual pyropheophorbide a dimer 5-4 targeting tumor acidic microenvironment was designed and synthesized,based on the acid-sensitive cis-aconitic acid linker and FRET theory,The pH-dependent fluorescence recovery rate in aqueous solution,combined with the potent in vitro PDT activity of 5-4 and its hydrolysate(IC50=1.37±0.13 and 0.27±0.01 ?M,respectively)indicated that the dimer model had the potential to be applied to tumor acidic microenvironment targeted fluorescence imaging and PDT treatment.