| Background and Objectives：Despite recent research involving cancer development and metastasis has gainedsignificant progress, malignant tumor still remains one of the most deadly diseases in theworld, due to the inefficient early diagnosis, poor specificity of drugs resulting in severeadverse effects, and lack of sensitive and real-time modalities to monitor therapeuticresponse. Tuomr theranostics, a fusion of therapeutics and diagnostics for optimizingefficacy and safety in cancer treatment, has been considered as a significant alternativenessto overcome these challenges. This integration can monitor therapeutic efficacy followingtreatments which can expedite clinician’s individualized therapeutic decisions. Becausetheranostic agents are of importance to achieve the simultaneous multifunctionality oftumor targeting, imaging and therapy, their preparation has received a great deal of researchinterest in recent years.Currently, there are two main strategies explored for the obtainment of suchmultifunctional theranostic agents. One approach is through the multi-step chemicalconjugation of anticancer drugs and contrast agents with various cancer-targeted ligands,such as small molecules, antibodies, peptides, aptamers, etc. With the rapid development onadvanced multifunctional nanomaterials in nanomedicine, the other approach fordevelopment of cancer theranostic agents is based on nanoplatforms. They achievesimultaneous cancer specific detection and therapeutics by specifically delivering a highlypotent cytotoxic agent toward tumors either through the EPR effect of the tumormicrovasculature or through the conjugation of target ligands which can specifically bind tobiomakers highly associated with cancer cells. In many cases, advanced nanomaterialsendowed with imaging capability have been engineered to deliver and release drugsselectively toward tumor tissues. With the two strategies referred abrove, some cancer targeted therapeutic drugs are approved for clinical use or clinical trial. For example,99mTclabeing lipidosomes loaded with DOX has been approved to carry out clinic Phase I trial. Itachieves treatment of head and neck squamous carcinoma by SPECT imaging.However, the conjugation may alter the functional activities of tumor-targeted ligands,contrast agents or therapeutic agents. Multi-step chemical conjugation may lead to lowerthe specificity of tumor ligands, weaken the imaging intensity of contrast agents, or lose theantitumor activity of drugs. Meanwhile, additional reaction agents and purificationprocedure in the multi-step conjugation would require higher cost. In particular,nanoplatform-based strategy for the multifunctional nanotheranostic agents has been provenchallenging and is still at an early or proof-of-concept stage due to several fundamentalproblems and technical barriers, such as the lag effect from reticuloendothelial system (RES)and mononuclear phagocytic system, the difficulties in large scale preparation with highreproductivity, and the potential safe concerns for their long-term fate and toxicity. Thus,investigation of new strategy for obtainment of theranostic agents is high necessary.Heptamethine cyanine dyes with two terminal indole heterocyclic units linked by apolymethine bridge, characterized with high molar absorption coefficient and fluorescencequantum yield, good photostability, have been applied widely in labeling nucleic acids andproteins, gene sequencing, in vivo imaging animals. Of them, Indocyanine green (ICG)has been used in clinic extensively for visualizing tiny blood vessels of livers and eyes.Recently, our studies have characterized a heptamethine cyanine dye, IR-780with tumortargeting and NIR imaging properties without chemical conjugation of tumor target ligand.The targeting property of IR-780also has been confirmed in a broad spectrum of tumorcells and tumor xenografts, suggesting an attractive diagnostic agent for sensitive andnoninvasive tumor detection. The subcellular localization of IR-780in tumor cells furthershowed that the dye exclusively accumulated in the mitochondria of tumor cells. Theunique property of delocalized lipophilic cation, may give IR-780the ability to target andretain in mitochondria of tumor cells.Based on our previous findings on IR-780simultaneously with tumor targeting andNIR imaging properties, we hypothesized:(1) whether IR-780could be used as a carrier tochemically conjugate antitumor drugs for tumor targeted imaging and therapy;(2) whetherthe intrinsically multifunctional heptamethine cyanine dyes could be obtained by rational modification of IR-780without need of chemical conjugation of antitumor durgs. If thesedesigns mentioned above are proved, they would provide a valuable strategy for preparationof theranositc agents applied in cancer personalized therapy.Methods and Results：In order to verify the hypotheses referred above, and obtain multifunctionalheptamethine cyanine dyes with tumor targeted imaging and therapeutic properties, thisdissertation is divided into three chapters and main results are as follows:1. Synthesis and characterization of a carboxyl derivative of IR-780for tumortargeting and NIR imaging1.1A modified synthetic method for heptamethine cyanine dyes was established. Dueto low efficiency and small-scale limitation in preparing heptamethine cyanine dyes aspreviously reported in literatures, an improved synthetic method needs to be established.The synthetic routes of heptamethine cyanine dyes including Vilsmeier-Haack formylationreaction, N-alkylation reaction and condensation reaction, were optimized for theconditions of reaction and post-treatment, resulted in the improvement of syntheticefficiency with large-scale available preparation, providing a basis for further drugconjugation.1.2IR-808, a derivative of IR-780with two reactive carboxyl groups was synthesized.In order to obtain multifunctional heptamethine cyanine dyes simultaneously with tumortargeted imaging and antitumor activites, IR-780needs to chemically conjugate with anantitumor drug. However, IR-780is lack of available functional group in its structure forfurther conjugation. IR-808was synthesized by using6-bromo hexanoic acid as the startingmaterial to introduce a carboxyl group in a key raw material which was used to synthesizeIR-780. Its structure was comfirmed with Hydrogen nuclear magnetic resonance (1H-NMR),Carbon-nuclear magnetic resonance (13C-NMR) and High resolution mass spectrum(HRMS).1.3IR-808was identified with good tumor targeting and NIR imaging properties.After determining the spectra of IR-808, it was found the peak wavelength of absorbtionand emission was in the NIR region (700-900nm). IR-808exhibited NIR fluorescentimaging ability and pretty good stability in serum. The preferential in vivo tumoraccumulation of IR-808was comfirmed in rTDMCs, HeLa and LLC tumor xenografts, suggesting a prospective potential used for tumor specific imaging and diagnosis. Insummary, our results showed that IR-808with two reactive carboxyl groups exhibited thetumor targeting and NIR imaging properties, providing a basis for further drug conjugation.This finding also provides a possibility in developing a radionuclide-labelled heptamethinecyanine dyes for deep-tumor imaging and diagnosis.2. Conjugation of IR-808with antitumor drugs for tumor-targeted imaging andtreatment2.1Three clinically available antitumor drugs were covalently conjugated with IR-808.On the basis of the above studies, IR-808was further designed to conjugate with antitumordrugs. We successfully conjugated IR-808with Melphalan,5-Fluoro-2,4(1H,3H)pyrimidinedione (5-Fu), Doxorubicin(DOX), and obtained IR-808NM, IR-808-5Fu andIR-808DOX, respectively. IR-808NM and IR-808-5Fu were afforded with high purity, andtheir structures were comfirmed with1H NMR and HRMS. Successful conjugation of DOXwith IR-808was ensured by the testing report of HRMS. However, IR-808DOX wasafforded with a low pure complex even through continuously optimizing the conditions ofreaction and purification.2.2The tumor targeted imaging and antitumor activity of IR-808NM and IR-808-5Fuwere investigated. The two conjugations were injected into the rTDMCs tumor-bearingmice to evaluate their specificity for tumor imaging. In vivo NIR imaging showed thatIR-808NM remained the tumor-targeted NIR imaging ability while IR-808-5Fu failed. Then,the antitumor activity of IR-808NM was evaluated with MG63osteosarcoma cancer cellsand SW480colon cancer cells. It was found that IR-808NM inhibited the growth of cancercells efficiently. Its antitumor activity was significantly higher than that of Melphalan.These results supported that we successfully developed a multifunctional heptamethinecyanine dye with chemical conjugation strategy, and verify the hypothesis that this kind oftumor targeting NIR dyes can be used as a carrier of antitumor drugs for tumor targetedimaging and therapy.3. Synthesis of esterified derivatives of IR-808for tumor treatmentAccording to our previous work and others, some caynine dyes, including IR-780would exhibit mitochondrial toxicity at higher concentration. Because mitochondria areencircled with two lipophilic membranes, lipophilic agents are more readily transported across bistratal membranes to reach a higher concentration. Basing on these investigations,we hypothesized that esterification of IR-808, would increase its lipophilicity greatly, aswell as its mitochondrial accumulation and potential toxicity. In this case, multifunctionalheptamethine cyanine dyes simultaneously with tumor targeted imaging and antitumoractivity would be obtained without need of chemical conjugation to tumor specific ligands.3.1Four esterified derivatives of IR-808were synthesized and characterized with NIRimaging property. In order to improve the lipophilicity of IR-808, and significantly enhancetheir accumulation and toxicity in tumor mitochondria, several esterified derivatives ofIR-808including butyl ester (IR-808DB), hexyl ester (IR-808DH), cyclohexyl ester(IR-808DCH), phenol ester (IR-808DP) were prepared with a highly efficient andlarge-scale available method. All these newly synthesized molecules have not been reportedpreviously. These molecules in methanol, DMSO and serum exhibited the maximumabsorption and emission wavelengths in the NIR region. Compared with ICG, esterderivatives of IR-808exhibited higher molar extinction coefficient and fluorescencequantum yields.3.2IR-808DB was characterized with significant antitumor effect. Cytotoxicity ofIR-808and its ester derivatives were performed on A549human lung cancer cells. Studiesshowed that these ester derivatives exhibited diverse anticancer activities on A549carcinoma cells. Of them, IR-808DB displayed most remarkable antitumor activity with anIC50of0.43μM. We further confirmed the antitumor effect of IR-808DB in other humancancer cell lines, including breast cancer cells (MDA231and MCF-7), hepatoma cells(SMMC-7721, HepG2), and non-small-cell lung cancer NCIH-460cells, demonstrating apotent antitumor activity of IR-808DB (IC50<6μM) on a broad spectrum of tumor cells. Invivo tumoricidal activities of IR-808DB were then evaluated in rTDMCs, A549, LLC, andHeLa tumor xenografts. Results revealed that5mg/kg IR-808DB inhibited tumor growthobviously as compared to20mg/kg Cyclophosphamide (CXT) group, a classic antitumordrug which has been widely applied in clinic. Laudable tumoricidal activities of IR-808DBwere observed in rTDMCs, A549, LLC and HeLa tumor xenografts, suggest the potentantitumor activity of IR-808DB in a variety of tumor xenografts. Meanwhile, the bodyweight and physical conditions of mice after treatment were not changed significantly. Inaddition, organs harvested from the mice with IR-808DB treatment were subjected to histopathological analysis and also showed no apparent abnormalities. The tumor growthinhibition of IR-808DB was also compared with IR-808NM, and results revealed thatIR-808DB displayed significantly higher tumor growth inhibition effect than IR-808NMdid.3.3IR-808DB was demonstrated with the ability of tumor-targeted imaging. In vivoNIR fluorescence imaging of athymic nude mice bearing with rTDMCs tumor xenograftsshowed IR-80DB with tumor targeting capability. The fluorescent intensity of the dissectedorgans and tumors further confirmed the preferential accumulation of IR-808DB in tumors.To evaluate its potential application in tumor early diagnosis,0.4mg/kg (imaging dosage)of IR-808DB was injected into the rats bearing rTDMCs tumors through tail vein and thefluorescent signals associated with tumor site were clearly detected even the tumor was notvisualized by eyes, indicating a potential used for tumor early identification and diagnosis.Conclusion and Innovation:In this dissertation, we initially established a modified synthetic method for IR-780and its derivatives, synthesized an analogue of IR-780with reactive carboxyl groups(IR-808) with tumor targeting and NIR imaging properties, providing a basis for furtherdrug conjugation. Next, IR-808was conjugated with three clinically available antitumordrugs respectively, and a multifunctional derivative of IR-808with tumor targeting andantitumor activity (IR-808NM) was successfully obtained. These results supported that wesuccessfully developed a multifunctional heptamethine cyanine dye with chemicalconjugation strategy, and verify the hypothesis that this kind of tumor targeting NIR dyescan be used as a carrier of antitumor drugs for tumor targeted imaging and therapy. Finally,by esterification of IR-808to increase its lipophilicity as well as its mitochondrialaccumulation and toxicity, we eventually obtained a multifunctional heptamethine cyaninedye (IR-808DB) intrinsically with tumor targeted imaging and antitumor effect withoutneed of chemical conjugation to tumor specific ligands. As a potential antitumor drug,IR-808DB has advantages of not only good tumor targeting, potent antitumor effect andfine NIR imaging, but also small molecular weight, low cost and large-scale availablepreparation. This multifunctional small molecule presents a valuable strategy forpreparation of new theranositc agents in cancer personalized therapy. |
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