Fabrication And Characterization Of Highly Fluorescent Carbon Quantum Dots Loaded Photosensitizer

Backgrounds:With the development of China's population and old-aging problem,cancers have been considered as one of the leading diseases resulting of people death.Hence,exploring a new technology to improve early diagnosis,real-monitoring and effective treatment of cancers is a key point to be solved.Photodynamic therapy?PDT?is one of the most promising routines in the fields of tumor detection and treatment.PDT has a lot of advantages such as high selectivity,non-invasion,few side effects and none drug-resistance.However,there remain exists some problems in the clinical applications.One is poor water solubility of photosensitizers?PS?,which causes the self-aggregation in water and low quantum yield of PS,the other is short penetration of light that limit the application of PDT in deep-seated tumors.Carbon quantum dots?CDs or C-dots?are a new group of zero-dimension carbon nanomaterials or nanoparticles,which have been demonstrated as the excellent nano-carriers of the drug due to their versatile preparation routes,better biocompatibility,hydrophilic and particularly tunable photoluminescence?PL?.Meanwhile,using the self-illumination system as the inner light,which hold a great potential to improve the limitation of PS and overcome the light penetration in deep lesions that are intractable by traditional PDT alone.Objective:Using high fluorescence and water soluble CDs as drug delivery of PS to fabricate a FRET system,which can significantly improve the limitation of PS.Meanwhile,the CDs can transfer the energy to the PS through FRET to increase the quantum yield of PS.Using the self-illumination as the inner light to activate the PDT system to enhance the short penetration of light and inefficient excitation of PS in vivo.Therefore,we aim to design a self-illumination induced PDT system with excellent biocompatibility,high quantum yield of PS,good water solubility,activation in vivo without an outer light,and finally to solve the limitation of PS and light in PDT applications and enhance the therapeutic effect.Methods:?1?The fluorescence CDs are synthesized by one-step hydrothermal method by using citric acid as carbon source and 1,2-ethylenediamine?EDA?as the passivation agent.The samples of CDs under different reaction conditions are prepared by changing the reaction temperature and reactant quantity.The properties of different CDs are evaluated by FT-IR,UV-Vis and fluorescence detection.Finally,the formation rules and fluorescence mechanism of CDs are estimated and summarized.?2?The Ce6loaded onto the CDs is fabricated using a modified EDC/NHS reaction,CDs are introduced magnesium metallic chelates to modify the CDs to further enhance the fluorescence intensity.Meanwhile,we attempt to take EDA as the surface passivation agents and connection agents between CDs and Ce6 to decrease the donor-acceptor distance and increase number of acceptors per donor to enhance the FRET efficiency as possible,the CDs-Ce6 conjugate is activated by a 660 nm laser,and the singlet oxygen detection and PDT assessment in vitro are performed to evaluate the therapeutic effect of tumor.?3?Design a bioluminescence?BLS?induced PDT system which is based on high luminescent and upconverted CDs,the as-prepared CDs connect with protoporphyrin IX?PIX?sensitizer to construct a PDT system and enable the excitation of the sensitizer with firefly luciferase induced BLS.The PDT system is evaluated by FT-IR,UV-Vis and fluorescence detection and the therapeutic effect of bioluminescence-induced PDT in vitro is also performed.?4?Bright yellow-emissive carbon quantum dots?y-CDs?are synthesized by an one-step solvothermal method using 1,2,4-triaminobenzene dihydrochloride as carbon source and formamide as solvent medium.Subsequently,luminol-H₂O₂-horseradish peroxidase?HRP?chemiluminescence?CL?,a potential inner light source,is explored to excite the y-CDs-Ce6 conjugate to overcome the penetration limit of excitation light.The Ce6 is designed to be excited efficiently by the CL through CRET and CRET-to-FRET two paths.Results:?1?CDs,preparation by one-step hydrothermal method using citric acid as carbon source and 1,2-ethylenediamine?EDA?as the passivation agent,had great PL,water solubility and biocompatibility.The as-prepared CDs were the excellent nano-carriers of drug delivery.The optimal reaction conditions were:citric acid 2.1 g,EDA 2.68 ml and the reaction temperature was 200 ^oC.The quantum yield of CDs were measured to be 79.7%.The fluorescence properties of CDs were determined by nuclei and surface states.The structure of CDs were similar to core-shell and its forming process mainly included the steps of cracking,polymerization,carbonization and growth of neclei.?2?Introducing magnesium metallic chelates to modify the CDs could further enhance the fluorescence quantum yield and the as-prepared CDs could covalent bonding with PS through EDC/NHS reaction.The CDs-Ce6 conjugate was successfully prepared with good solubility,and the fluorescence of CDs-Ce6 conjugate presented a stronger fluorescence,about 10-folds than that of pure Ce6.At the same time,the FRET efficiency in CDs-Ce6 conjugate was increased to 84%by decreasing donor-acceptor distance and increasing number of acceptors per donor,the singlet oxygen production of CDs-Ce6 was twice larger than that of Ce6.Increment of FRET efficiency could significantly improve the therapeutic effects in PDT.?3?BLS instead of external illumination source was used to activate designed CDs-PIX conjugate.The upconverted CDs acted an important role in CDs-PIX conjugate,including improve the solubility,enhanced the fluorescence of PIX through FRET and regulated the BLS to meet the qualification for PIX.Results indicated the doped PIX could be excited by the BLS,producing enough singlet oxygen?SO?in vitro and revealing an efficient therapy for SMMC-7721 tumor cells killing?about 60%?through FRET process.?4?The y-CDs,preparation by a solvothermal method using 1,2,4-triaminobenzene dihydrochloride as carbon precursor,exhibited excellent dispersibility and solubility.The PL excitation spectrum was ranging from 400-550 nm,which was well overlapped with CL spectrum.In this design system,the nano-carrier of y-CDs,the potential internal light of CL and the PDT agent of Ce6 were carefully selected and united to form an efficient and cooperating system.The y-CDs played double functional roles:nano-carrier of PS to modify its hydrophilicity and light adjuster to mediate the internal light to activate the Q-bands of Ce6,and two main absorptions of Ce6?400 and 663 nm?in y-CDs-Ce6conjugate could be activated efficiently by the CL through direct CRET and indirect CRET–FRET,which brought with a high yield of reactive oxygen species?ROS?and a remarkable efficiency of 92%in hepatoma cells?SMMC-7721?killing,and these results were much higher than that of Ce6 alone.Conclusion:?1?CDs have good water solubility,high fluorescence quantum yield,simple synthesis process and excellent biocompatibility,which are proved to be a superior nano-carrier of the PS.?2?CDs conjugation with PS can significantly improve dispersibility and solubility and enhance the fluorescence performance of PS through FRET.Increment of FRET efficiency can significantly improve the therapeutic effects in PDT,which may have great potential for enhanced applications in tumor treatment.?3?BLS and CL can be used as the inner light to activate the modified photosensitizer to produce reactive oxygen species and complete the PDT treatment.This designed self-illumination induced PDT system hold a great potential in overcoming light penetration issues and treating deeper lesions that are intractable by traditional PDT alone.