| Background and objectives:Cancer phototherapy has become one of main means for cancer treatment available in clinic following with surgical treatment,chemotherapy and radiotherapy.During the treatment,tumor tissue selective photosensitizer is first administrated.And then irradiates tumor area with a laser at wavelengths corresponding to the absorption spectrum of the photosensitizer.So that the photosensitizer efficiently catalyzes oxygen molecules into reactive oxygen species(ROS)which will damage cell structure or function,or convert the absorbing light energy into local hyperthermia,and eventually achieve the antitumor therapeutic effects.As comparing to the major current clinical treatments for cancer involving surgery,chemotherapy,radiotherapy,Phototherapy possess small lesion,high selectivity and good safety,is expecting to play an important role in cancer therapy.According to the different therapeutic mechanisms,Cancer phototherapy,mainly involving photodynamic therapy(PDT)and photothermal therapy(PTT).Either for PDT or PTT,the key factor to determine their therapeutic effects is to develop the desirable photosensitizer.An ideal PS should have efficient phototherapeutic outcome with few side effects,which requires it possess these two characteristics: superior photosensitivityand high specificity upon cancer cells.(1)In order to improve the photosensitivity:On the one hand,a lot of well-designed nanomaterials have been fabricated by incorporation of a photothermal PS and a photodynamic PS for synchronic PDT and PTT treatment,greatly improving the therapeutic effect.On the other hand,since the absorption of biological tissue is weak in the near infrared region(700-900nm),so choose a near infrared photosensitizer will achieve good photo-therapeutic effect for deeper tumor,which greatly promoted the prospect of optical treatment.(2)To enhance the specificity,the most common strategy is chemical conjugation of PSs with target ligands,which can efficiently bind to biomarkers over expressed on the membranes of cancer cells.However,nanomaterial-based multifunctional PSs will confront with many fundamental problems,such as difficulties in large-scale preparation with good repeatability,accumulation in reticuloendothelial organs,toxicity concerns for the long-term fate,leading that most of them are still on an early stage of development.Meanwhile,development of multifunctional PSs by multi-step chemical conjugation may alter the structures of target ligands,mean additional synthetic steps and costs,which also hinder their practical application.In view of the above shortcomings of conventional photosensitizers,it is critical to developsmall-molecule PSs by their structure-inherent multifunctionality without conjugation.These small-molecule PSs are expected to avoid these shortcomings,and greatly improving the potential clinical applications of cancer phototherapy further promoting its prospects.In the previous studies,we have synthesized and identified a class of heptamethine cyanine dyes inherently with preferential tumor accumulation without chemical conjugation of target ligands.They retain selectively in a broad-spectrum of cancer cells and accumulate at mitochondria.Mitochondria are the vital organelles for cell survival as they play a central role in energy production and apoptotic pathways.Hence,mitochondria have been long recognized as the subcellular target suitable for cancer treatment.In particular,as mitochondria are susceptible to excessive ROS(such as singlet oxygen and free radicals)or hyperthermia,a number of PSs have been designed and demonstrated with improvement of PDT or PTT therapeutic effect by mitochondria targeting.In addition,certain cyanine dyes have been demonstrated with photosensitization effects.Based on these findings,we hypothesize that a NIRheptamethine cyanine-PS integrating PDT and PTT treatment by targeting mitochondria will further improve the phototherapeutic effect.In addition,due to the easy synthesisthese small-molecule photosensitizerspossess more potential for clinical applications.Methods and ResultsOur structure-activity relationship study and other reports indicate that the heptamethine core with lipophilic cationic property is essential for cancer-cellmitochondria targeting and imaging.In addition,certain cyanine dyes have been demonstrated with photosensitization effects,which are closely associated with their lipo-hydro partition coefficients(Log P),polarizabilities,molar absorption coefficients.In order to verify the hypotheses and obtain multifunctional PSs with mitochondria-targeted and PDT/PTT combinational treatment,this study is divided into three chapters and main results are showed as follows:1.Design and chemical synthesis of mitochondria-targeted small-molecular photosensitizers: Based on our structure-activity relationship study,a series of heptamethine cyanine dyes by modification of N-alkyl side chains based on the mitochondria-targetedheptamethine core were designed and synthesized.They were varied not only with different chemical groups at the end of side chains(including methyl,methoxyl,aromatic,hydroxyl,sulfo,carboxyl groups,etc.),but also with different length of alkyl chain around the heptamethine core(redcircle in Scheme 1,n=1-5).The modifications render them with variable Log P,polarizability and molar absorption coefficient,providing potential for developing a mitochondria-targeted small-molecule PS synchronously with both PDT and PTT effects.2.The screening of target photosensitizer and its’ in vitro PDT/PTT therapeutic effect and mechanism study: After synthesis of 27 representative near infrared small molecules,their tumor targeting(accumulation difference between tumor tissue and muscle tissue),photothermal characteristics,photodynamic properties(generation of singlet oxygen and ROS levels)were evaluated.Finally,compound 7,a small molecular PS simultaneously with cancer-cell mitochondrial targeting,NIR imaging and synchronous PDT/PTT effects was screened out.Its high phototherapeutic efficacy was verified on A549,H460,MCF-7 and 4T1 cancer cells.The synergetic PDT and PTT effect of 7 was also verified by pretreatment with ice incubation(single PDT treatment)or pretreated with N-acetylcysteine(NAC,an effective scavenger for wiping out ROS for single PTT treatment).The mitochondrial localization of 7 was confirmed by co-staining with two kinds of mitochondrial tracker(Mito-tracker,Rho123),respectively.We next investigated the mechanism of photo-induced cytotoxicity through flow cytometry and western blot,indicating that cell apoptosis mediated by the intrinsic mitochondrial pathway plays an important role for its’ excellent phototherapeutic effect.3.In vivo PDT/PTT therapeutic effects of compound 7 on different animal xenograft models: In vivo phototherapeutic efficacy of 7 was initially elucidated in subcutaneous and orthotopic tumor xenograft models.Using NIR imaging,tumor preferential accumulation of 7 was demonstrated in three tumor xenograft models above.After NIR light irradiation(808 nm,0.8 W.cm-2,5min),both the hyperthemia and ROS generated in tumor tissues were detected,further demonstrating the in vivo synergetic PDT and PTT effect.After irradiation,tumor sizes and survival rates were observed.Tumor growth in the 7 irradiation groups was completely inhibited,and no tumor recurrence was observed with 100% survival rate during the observation period.Blood routine and renal/liver function parameters of mice in each group was monitored during treatment.Results showed that there was no statistical significant difference between treated group and control group,suggesting the well-tolerance and unobvious photo-induced side effects of 7.In addition,we further confirmed theexcellent phototherapeutic efficacy of 7 on patient-derived xenograft models to illustrate the potential for clinical utility.ConclusionIn conclusion,a small molecular PS termed as compound 7 simultaneously with cancer-cell mitochondrial targeting,NIR imaging and synchronous PDT/PTT effects was developed in this study.Its’ targeted NIR imaging property makes tumors with the margins clearly visualized,which is greatly helpful for imaging-guided phototherapy and real-time treatment monitoring.To our knowledge,this is the first report that a small-molecule photosensitizer integrates both cancer PTT and PDT treatment by targeting mitochondria.This photosensitizer possess excellent photosensitivity,is expected to improve the phototherapeutic effect with minimal side toxicity further providing a practical way for cancer treatment. |
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