Preparation And Properties Of Carboxylesterase-activatable Theranostic Prodrug Nanosystem

With the rapid development of chemistry,nanotechnology,pharmacy,biotechnology and imaging technology,a large quantity of novel drug delivery systems(DDS)have been designed and fabricated.Compared with the direct use of drug for traditional chemotherapy,utilizing DDS to deliver drugs can alleviate side effects to normal tissues and enhance therapeutic efficiency.In particular,theranostic DDS which combines the specificity toward tumor tissues with fluorescence imaging draw a lot of attention.They can not only be activated by endogenous or exogenous substances,but also can monitor and track the release of drug with cell imaging.In this paper,we firstly designed and synthesized a novel carboxylesterase-based(CaE-based)theranostic prodrug.This prodrug comprises three parts: 2-OH-coumarin as the fluorogen and cleavable architecture,chlorambucil(CBL)as the anticancer drug,and ethylester group as the enzyme-responsive unit and can quench the fluorescence of 2-OH-coumarin via intramolecular charge transfer(ICT).To further alleviate the side effects and prolong the circulation time for better therapeutic efficiency,by taking full advantage of amphilic polymers,we incorporated the prodrug into liposomes to obtain the liposome drug delivery systems.After the uptake by HeLa cells,the DDS will dissociate,the prodrug enters into cytoplasm and upon activation by the enzyme CaE,due to the cleavable reaction,the active drug and the fluorophore are released simultaneously;the ICT effect is diminished,and the fluorophore's fluorescence is restored.Through the change of fluorescence,we can monitor the release of drug.This DDS plays a double role of diagnosis and treatment.The main contents and results in this dissertation are as follows:The structures of intermediates and the target compound were characterized and confirmed by high resolution mass spectrometry,1H-NMR and 13C-NMR.The prodrug was incorporated in liposomes and the shape and distribution of liposome nanoparticles were observed: nanoparticles are spherical with a uniform diameter of about 100 nm.We also synthesized the control to validate the mechanism of action.Through a series of tests in vitro,we investigated the release behavior,stability and specificity.The main results include: this DDS has an excellent specific response toward CaE,and will not be influenced by other bio-substances.The release of CBL and fluorogen 2-OH-coumarin was further confirmed by HPLC and HRMS.Via the change of fluorescence intensity,we can monitor the extent of drug release.The prodrug has the potential for quantitative detection of CaE.Meanwhile,the prodrug is stable towards different pH levels and the release of drug will not be affected by pH change.From the results from cell experiment conducted by HeLa cells,we can know that the CBL can be released after the uptake of DDS by HeLa cells with the fluorescence signal increased;cell toxicity and apoptosis assays show that this DDS has a predominant inhibition towards cell proliferation.In summary,the novel CaE-based theranostic prodrug we designed and synthesized can realize real-time visualization of drug release processe and show an excellent selectivity towards CaE.The DDS can also alleviate the side effects to normal tissues and enhance the therapeutic efficiency.This DDS is supposed to provide some helpful insights for the design and application of theranostic prodrugs.