| Gemcitabine(GEM)is a potent chemotherapeutic agent of nucleoside analogs aginst a wide range of solid tumors.Notably,GEM has been the first line and the gold standard drug for metastatic or advanced pancreatic cancer till now.However,the clinical application of GEM is greatly limited by its intrinsic deficiencies,such as very short plasma half-life,poor membrane permeability,lack of tumor-specific distribution and so on.Up until now,prodrugs and nanomedicines have been two main technologies in GEM delivery and have already made some achievements.However,small molecular prodrugs are always lack of tumor targeting ability,and nanomecicines are faced with formidable challeges towards clinical translation.So it’s urgent to develop new drug delivery systems for better application of GEM.In situ macromolecular prodrug strategy,namely,in situ covalent-albumin-binding prodrug strategy was exploited here.We attempted to overcome various physiologic barriers in GEM delivery by use of endogenous albumin as a drug carrier.A cathepsin B-responsive GEM prodrug(GAM)was constructed first.To improve its undesired controlled release profile,four reduction-responsive prodrugs(8a,8b,11a,11b)were then constructed exploiting different controlled release methods.We were aiming at screening out the prodrug with best controlled release profile as well as with the best antitumor efficacy.At the same time,in the research of reduction-sensitive prodrugs,theoretical foundation was made through rating the benefits and risks of different combinations of linkage bond/linker to controlled release profile and therapecutic outcomesCTB-responsive prodrug(GAM)bound GEM with maleimide group by a peptite bond-containing linker.The reduction-sensitive prodrugs were divided into two groups:(a)carbonate formation at the 5’-OH group of the sugar moiety and(b)carbamate masking the 4-NH2 group of the cytosine base.In each group,GEM was bound through an all-carbon or self-immolative disulfide bond-containing linker to maleimide group.We designed and synthesized the five prodrugs successfully,and their structures were characterized by HRMS,1HNMR和 13CNMR.All prodrugs were capable of rapid and specific binding of Cys-34 from bovine serum albumin(BSA)or endogenous albumin from rat blood.The albumin-prodrugs were also characterized by HRMS.In the plasma stability test and cytidine deaminase(CDA)resistance test,five albumin-prodrug conjugates manifested good plasma stability and degradation resistance aganist CDA.As to the reduction-sensitive prodrugs,the more stable the combination of linkage bond/linker was,the more stable the albumin-prodrug conjugates against CDA would be.Next the in vitro drug release profiles of the two kinds of prodrugs were investigated.Compared with non-CTB-responsive GCM,GAM showed good CTB respond and greatly accelerated GEM release rate in simulated lysosome environment.However,GAM only released less than 60%of total GEM after 72 h incubation.In the research of reduction-sensitive prodrugs,compared with disulfide bond-lacking 8a and 11a,disulfide bond-containing 8b and 11b released GEM in a significantly accelerated way in simulated intracellular reductive environment;but they also divulged GEM more in simulated extracellular oxidative environment.These results demonstrated that the introduction of a disulfide bond would bring about burst drug release in the tumor and unfavorable pharmacokintic profile at the same time for a prodrug.We studied the cytotoxicity,inracellular GEM release,cell uptake and hENT dependence of 5 free prodrugs or their albumin conjugates on 4T1 cells.Stimuli-responsive prodrugs or their conjugated form exibited stronger cytotoxicity and faster intracellular GEM release when compared with their non-stimuli-responsive counterparts.In addition,the cell uptake of 5 kinds of BSA-prodrug conjugates was in an albumin-dependent and hENT-independent active-transport process,implying opportunities to circumvent acquired multidrug resistance caused by decreased expression of hENT.In Sprague-Dawley(SD)rats,the pharmacokinetic profiles of 5 prodrugs were investigated.All the prodrugs in the body mainly existed in macromolecular prodrug form and released GEM in a much slower and less way,resulting in dramatically decreased CDA deactivation.As to the reduction-sensitive prodrugs,when compared with GEM,the bioavailability of 8a and 8b enhanced 21-fold and 7-fold respectively;the plasma half-life of 8a increased 3.6-fold while that of 8b did not show obvious increase.These data suggested that the extent of improvement in pharmacokinetic profile of a prodrug was in positive correlation with the stability of the combination of linkage bond/linker and that disulfide bond introduction brought about instability in circulation and unfavorable pharmacokinetic profile.In 4T1 tumor-bearing mice,we investigated the in vivo biodistribution of albumin-prodrug conjugates labelled by Cy5.The prodrug conjugates demonstrated stronger accumulation in the tumor and sparing much less distribution in normal tissues and organs especially in the kidney,liver and spleen.Finally,the in vivo antitumor efficacy of 5 prodrugs were investigated in 4T1 tumor-bearing mice.As to the CTB-sensitive prodrug group,the vehicle and non-CTB-responsive GCM couldn’t inhibit tumor growth effectively and GEM could inbibit tumor growth to some extent.Instead,GAM showed much better antitumor efficacy than GEM did and dramatically slowed down tumor progression with final tumor volume being only once bigger.The result indicated that CTB-responsive prodrug improved antitumor efficacy by promotion of tumor site-specific drug release.As to the reduction-responsive prodrugs,with tumor inhibition rate being over 90%,the carbonate group 8a and 8b were more potent in efficacy than the carbamate group 11a and 11b.Surprisingly,8a standed out as the most potent one and shrinked the tumor volume to 1/3.Compared with 11a,11b demonstrated accelerated tumor GEM release and then significantly enhanced antitumor efficacy;compared with 8a,8b manifested worse pharmacokenetic profile and then undermined therapeutic outcomes.As such data suggested that disulfide bond introduction would not always mean better controlled release profile and that the linkage bond between the parent drug and the linker could be the determinant.Better efficacy would be.obtained when the linkage bond and the linker are well coordinated.Moreover,all the mice either from the control group or prodrug-given group showed no significant body weight loss or liver and kidney toxicity.All above results indicated that GAM,8a and 8b were potent and safe drug delivery systems. |
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