Construction Of Triterpenoid Supramolecular Photosensitive Drugs And Their Antitumor Photochemotherapy Research

Supramolecular assembly,as a novel strategy for building smart nanodrugs,has made anticancer nanodrugs show great potential in clinical translation due to its simplified nanofabrication process,controlled drug activity,and physiotherapeutic function.Based on this strategy,supramolecular photosensitizers-mediated photodynamic therapy（PDT）has shown great promise for anticancer therapy because of its non-invasive,low toxicity,and high efficiency.However,the currently developed functional platforms based on supramolecular photosensitizers are generally incompatible in biocompatibility,simple and rapid construction（self-assembly）,biodegradability,and synergistic anticancer,which greatly limit the possibility of efficient clinical applications.Hence,it is of significance to develop a multifunctional single-component drug delivery platform that possesses the above properties simultaneously for the construction of clinically translatable photosensitive drugs.Whereas owing to the novel self-assembly ability,triterpenoid natural small molecules with anticancer activity showed hung advantages for the rapid construction of bio-safe and synergistic antitumor chemotherapeutic drugs.Based on the above consideration,this thesis takes self-assembled triterpene small molecules as the main research object and uses them as single-component carriers to prepare supramolecular photosensitizers for synergistic antitumor chemotherapy and PDT.The following three aspects were investigated systematically including putting forward the idea of triterpene-mediated photosensitive drugs,solving the defects of most triterpenoids as drug carriers using co-assembly strategy,and developing high-efficient but low toxic triterpenoid photosensitive prodrugs potentiate cancer immunotherapy.Firstly,the idea of using triterpenes as single component carriers for the preparation of supramolecular photosensitized drugs was proposed,and tetracyclic triterpene sterols with suitable self-assembled morphologies and sizes were used as the representative substances to explore the potential of self-assembled active triterpenoids in synergistic antitumor photochemotherapy.After screening the self-assembled morphology and anticancer activity of ergosterol（Ergo）,β-sitosterol,and stigmasterol,as well as their photo-toxicity after encapsulation of Chlorin e6（Ce6）,Ergo was selected as a representative to prepare photosensitive drugs Ergo-Ce6 NPs for systematic evaluation of the feasibility and effectiveness of triterpene sterols in constructing supramolecular photosensitizers.Using an emulsification method,Ce6 was successfully encapsulated in assembled Ergo-Ce6 NPs and existed in the monomer state via intermolecularπ-πstacking and hydrophobic interactions.Despite the lower Ce6 loading efficiency（3.4%）,the resultant Ergo-Ce6 NPs have enhanced reactive oxygen species（ROS）generation（1.4times compared with Ce6）by inducing type I photoreactions to significantly promote the·OH generation.Due to the significant in vitro anticancer synergistic effect of Ergo and Ce6 PDT（combination index CI=0.46）,as well as the excellent tumor accumulation,better biocompatibility,and efficient in vitro cytotoxicity of Ergo-Ce6 NPs（73%4T1 and92%MCF-7 cancer cells mortality at 1μg/m L of Ce6）,and in vivo antitumor inhibition efficiency of 86.4%was finally achieved.This work fully confirmed the feasibility and potential value of self-assembled triterpene compounds for constructing supramolecular nanomaterials.Secondly,the problem of mismatch in function,morphology,and activity of terpenoid natural small molecules（NSMS）as drug delivery carriers was addressed successfully by employing the co-assembly strategy.After screening,we identified 11compounds（triterpenes and diterpenes）that could form nanoscale co-assembled NSMs-Ce6 NPs with regulatable drug sizes and morphology.Analysis of the representative betulonic acid（BC）-mediated nano-coassemblies（BC-Ce6 NPs）revealed the high efficiency of the co-assembly strategy and highlighted the tremendous potential of NSMs as novel drug delivery platforms.Using the green and simple one-step reprecipitation method,the Ce6 loading efficiency successfully increased to 17.6%.Comparative analysis of molecular dynamics simulations of the pentacyclic triterpene GA/UA,the derivative UASS and UASS/Ce6 co-assemblies,confirmed that the linear coplanar arrangement structure of BC dimeric units was primarily responsible for the formation of rod-like BC self-assemblies,showing a difference in the morphology with spherical BC-Ce6 NPs.Through the potentialπ-πstacking between BC and Ce6,the reduced energy gap between the singlet and triplet excited states(ΔE_ST)of Ce6（from 1.2001 e V to 1.1980e V）facilitated efficient ROS generation（1.3 times compared with Ce6）of BC-Ce6 NPs by promoting·OH generation via a type I photoreaction mechanism.The assembled nanodrugs exhibited multiple favorable therapeutic features,such as high-efficiency synergy,improved water solubility,and ROS generation,enhanced tumor accumulation,thus ensuring a remarkably enhanced,synergistic,and secure combinatorial anticancer efficacy of 93.6%with highly efficient tumor ablation.Finally,to take full of the advantages of triterpenes in synergistic anticancer such as biosafety,simple nano-functionalization,while addressing the common deficiencies of most triterpene molecules with insignificant chemotherapeutic activity,and combining the lower anti-tumor immune response of clinical immunotherapy.Betulinic acid（BA）with relatively better anticancer activity and self-assembly performance was used as the representative of triterpenoids,and then we designed a novel carrier-free photo-chemotherapeutic nano-prodrug COS-BA/Ce6 NPs by combining additional two multifunctional components-a water-soluble chitosan oligosaccharide（COS）and a low toxic photosensitizer Ce6.The aim is to maximize the anticancer efficacy of BA nano-agents while minimizing its toxicity to normal tissue,and further to augment the antitumor efficacy of immune adjuvant anti-PD-L1-mediated cancer immunotherapy.Specially,we showed that the synthesis of the amphiphilic molecule COS-BA could significantly enhance the chemotherapeutic activity of BA.Compared with BA（17.63μg/m L）,COS-BA showed a lower IC₅₀ of 6.03μg/m L against 4T1 cancer cells.Meanwhile,theπ–πstacking between COS-BA and Ce6 might reduce the energy level difference of the single-triplet excited state of Ce6,thus promoting the intersystem crossing（ISC）leading to increased ¹O₂ generation（26%）of COS-BA/Ce6 NPs.More importantly,the engineered nanodrugs harbored a smart chemotherapeutic dormancy function that showed remarkably suppressed in vitro cytotoxicity regardless of against cancer or normal cells.Attributing to the distinctive p H-responsive behavior,it performed low-toxicity and bio-safety to normal tissue,but highly toxicity to tumor tissue owing to the stimulating release of hyperactive amphiphilic prodrug species in the weakly acidic tumor microenvironment.Moreover,when combined with anti-PD-L1 therapy,both nano-coassemblies based chemotherapy and chemotherapy/PDT could effectively activate antitumor immunity in treating primary or distant tumors by inducing robust CD⁸⁺cytotoxic T lymphocytes infiltration,which is anticipated to open up attractive possibilities for clinical immunotherapy.