title: Enzyme-activated nanoconjugates for tunable release of doxorubicin in hepatic cancer cells.
abstract: We report the synthesis of a series of aromatic azo-linkers (L1-L4), which are selectively recognized and cleaved by azoreductase enzymes present in the cytoplasm of hepatic cancer cells via a NADPH-dependent mechanism. We utilized L1-L4 azo-linkers to conjugate doxorubicin to generation 5 (G5) of poly(amidoamine) dendrimers to prepare G5-L(x)-DOX nanoconjugates. We incorporated electron-donating oxygen (O) or nitrogen (N) groups in the para and ortho positions of L1-L4 azo-linkers to control the electronegativity of G5-L(x)-DOX conjugates and investigated their cleavage by azoreductase enzymes and the associated release of loaded DOX molecules. Hammett σ values of G5-L(x)-DOX conjugates ranged from -0.44 to -1.27, which is below the reported σ threshold (-0.37) required for binding to azoreductase enzymes. Results show that incubation of G5-L1-DOX (σ = -0.44), G5-L2-DOX (σ = -0.71), G5-L3-DOX (σ = -1.00), and G5-L4-DOX (σ = -1.27) conjugates with human liver microsomal (HLM) enzymes and the S9 fraction isolated from HepG2 hepatic cancer cells results in release of 4%-8%, 17%, 60%, and 100% of the conjugated DOX molecules, respectively. These results show that increasing the electronegativity (i.e. lower σ value) of L1-L4 azo-linkers increases their susceptibility to cleavage by azoreductase enzymes. Intracellular cleavage of G5-L(x)-DOX nanoconjugates, release of conjugated DOX molecules, and cytotoxicity correlated with conjugate's electronegativity (σ value) was investigated, with G5-L4-DOX conjugate exhibiting the highest toxicity towards hepatic cancer cells with an IC50 of 13 nm ± 5 nm in HepG2 cells. Cleavage of G5-L(x)-DOX conjugates was specific to hepatic cancer cells as shown by low non-specific DOX release upon incubation with non-enzymatic insect proteins and the S9 fraction isolated from rat cardiomyocytes. These enzyme-activated G5-L(x)-DOX conjugates represent a drug delivery platform that can achieve tunable and cell-specific release of the loaded cargo in hepatic cancer cells.
authors.affiliation: University of Michigan, Department of Biomedical Engineering, Cellular Engineering and Nano-Therapeutics Laboratory, Ann Arbor, MI 48109, USA.
authors.forename: Scott H
authors.initials: SH
authors.lastname: Medina
authors.forename: Maxim V
authors.initials: MV
authors.lastname: Chevliakov
authors.forename: Gopinath
authors.initials: G
authors.lastname: Tiruchinapally
authors.forename: Yasemin Yuksel
authors.initials: YY
authors.lastname: Durmaz
authors.forename: Sibu P
authors.initials: SP
authors.lastname: Kuruvilla
authors.forename: Mohamed E H
authors.initials: ME
authors.lastname: Elsayed
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000008:administration & dosage* / Q000494:pharmacology / Q000633:toxicity; D002478:Cells, Cultured; D003692:Delayed-Action Preparations / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D050091:Dendrimers / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D004317:Doxorubicin / Q000008:administration & dosage* / Q000494:pharmacology / Q000633:toxicity; D056945:Hep G2 Cells; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000473:pathology; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000473:pathology; D032383:Myocytes, Cardiac / Q000187:drug effects; D058726:Nanoconjugates / Q000737:chemistry* / Q000633:toxicity; D011073:Polyamines / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D051381:Rats
chemical_list: D000903:Antibiotics, Antineoplastic; D003692:Delayed-Action Preparations; D050091:Dendrimers; D058726:Nanoconjugates; C531249:Poly(amidoamine); D011073:Polyamines; D004317:Doxorubicin