Aptamers Entirely Built from Therapeutic Nucleoside Analogues for Targeted Cancer Therapy.

Owing to the specific and high binding affinity of aptamers to their targets, aptamer-drug conjugates (ApDCs) have emerged as a promising drug delivery system for targeted cancer therapy. However, in a conventional ApDC, the aptamer segment usually just serves as a targeting moiety, and only a limited number of drug molecules are sequentially conjugated to the oligonucleotide, giving a relatively low drug loading capacity. To address this challenge, herein we employ four clinically approved nucleoside analogues, including clofarabine (Clo), ara-guanosine (AraG), gemcitabine (Ge), and floxuridine (FdU), to replace all natural nucleosides in aptamer sequences, generating a series of whole drug-constituted DNA-like oligomers that are termed drugtamers. Similar to their parent aptamers, the obtained drugtamers maintain the targeting capability and can specifically bind to the target receptors overexpressed on the cancer cell surface. With 100% drug loading ratio, active targeting capability, and enzyme-mediated release of active therapeutics, our drugtamers can strongly induce the apoptosis of cancer cells and inhibit the tumor progression, which enables a new potential for a better targeted cancer therapy.

Construction of synergistic pH/H2O2-responsive prodrug for prolonging blood circulation and accelerating cellular internalization.

We have developed a real-time and multifunctional doxifluridine-conjugate prodrug (LYX), which involved the preliminary methylfluorescein with 5-fluorouracil linker as protecting group, the targeting biotin unit, and a model therapeutic drug (doxifluridine). The shielding group (5'-DFUR) was found to be effective in prolonging circulation at physiological pH 7.4 and improving accumulation in the acidic microenvironment of the tumor. Based on this strategy, the stability and stimulus responsive properties of prodrug could enhance drug release efficiency and exhibit fewer side effects, thereby providing a unique opportunity for diagnosis and imaging additional analytes or enzymatic activities.

Evaluation of Floxuridine Oligonucleotide Conjugates Carrying Potential Enhancers of Cellular Uptake.

Conjugation of small molecules such as lipids or receptor ligands to anti-cancer drugs has been used to improve their pharmacological properties. In this work, we studied the biological effects of several small-molecule enhancers into a short oligonucleotide made of five floxuridine units. Specifically, we studied adding cholesterol, palmitic acid, polyethyleneglycol (PEG 1000), folic acid and triantennary N-acetylgalactosamine (GalNAc) as potential enhancers of cellular uptake. As expected, all these molecules increased the internalization efficiency with different degrees depending on the cell line. The conjugates showed antiproliferative activity due to their metabolic activation by nuclease degradation generating floxuridine monophosphate. The cytotoxicity and apoptosis assays showed an increase in the anti-cancer activity of the conjugates related to the floxuridine oligomer, but this effect did not correlate with the internalization results. Palmitic and folic acid conjugates provide the highest antiproliferative activity without having the highest internalization results. On the contrary, cholesterol oligomers that were the best-internalized oligomers had poor antiproliferative activity, even worse than the unmodified floxuridine oligomer. Especially relevant is the effect induced by palmitic and folic acid derivatives generating the most active drugs. These results are of special interest for delivering other therapeutic oligonucleotides.

Supramolecular Amphiphiles Based on Pillar[5]arene and Meroterpenoids: Synthesis, Self-Association and Interaction with Floxuridine.

In recent years, meroterpenoids have found wide biomedical application due to their synthetic availability, low toxicity, and biocompatibility. However, these compounds are not used in targeted drug delivery systems due to their high affinity for cell membranes, both healthy and in cancer cells. Using the approach of creating supramolecular amphiphiles, we have developed self-assembling systems based on water-soluble pillar[5]arene and synthetic meroterpenoids containing geraniol, myrtenol, farnesol, and phytol fragments. The resulting systems can be used as universal drug delivery systems. It was shown by turbidimetry that the obtained pillar[5]arene/synthetic meroterpenoid systems do not interact with the model cell membrane at pH = 7.4, but the associates are destroyed at pH = 4.1. In this case, the synthetic meroterpenoid is incorporated into the lipid bilayer of the model membrane. The characteristics of supramolecular self-assembly, association constants and stoichiometry of the most stable pillar[5]arene/synthetic meroterpenoid complexes were established by UV-vis spectroscopy and dynamic light scattering (DLS). It was shown that supramolecular amphiphiles based on pillar[5]arene/synthetic meroterpenoid systems form monodisperse associates in a wide range of concentrations. The inclusion of the antitumor drug 5-fluoro-2'-deoxyuridine (floxuridine) into the structure of the supramolecular associate was demonstrated by DLS, 19F, 2D DOSY NMR spectroscopy.

Hypoxia-Activated and Indomethacin-Mediated Theranostic Prodrug Releasing Drug On-Demand for Tumor Imaging and Therapy.

A smart theranostic prodrug IMC-FDU-TZBC-NO2, releasing active drug on-demand based on hypoxia-activated and indomethacin-mediated, for solid tumor imaging and efficient therapy was designed. This prodrug was constructed by conjugating chemotherapy drug 5-fluoro-2-deoxyuridine (FDU), targeting moiety indomethacin (IMC), and the hypoxic trigger 4-nitrobenzyl group to a fluorescent dye precursor, which was mediated by IMC and activated by NTR under hypoxic conditions. The fluorescent dye IMC-TZBCM was generated and FDU was released at the same time in tumor cells. The rates and amounts of FDU release and IMC-TZBCM generation were regulated by hypoxia status, and increased with increasing degree of hypoxia. Nevertheless, it is "locked" in normal cells. It combined the advantages of tumor targeting, diagnosis, and chemotherapy functions, showed excellent targeting ability to cancer cells, excellent stability in physiological conditions, high cellular uptake efficiency, and on-demand drug release behavior. The in vitro and in vivo assays demonstrated that IMC-FDU-TZBC-NO2 exhibits enhanced anticancer potency and low side effects. The novel targeted theranostic prodrug activated by hypoxia shows a great potential in cancer therapy.

Prototyping kinase inhibitor-cytotoxin anticancer mutual prodrugs activated by tumour hypoxia: A chemical proof of concept study.

Amide- and ester-linked kinase inhibitor-cytotoxin conjugates were rationally designed and synthesised as prototype hypoxia-activated anticancer mutual prodrugs. Chemical reduction of an aryl nitro trigger moiety was shown to initiate a spontaneous cyclisation/fragmentation reaction that simultaneously released the kinase inhibitor semaxanib (SU5416) and the amine- or alcohol-linked cytotoxin from the prodrugs. Preliminary cell testing and reduction potential measurements support optimisation of the compounds towards tumour-selective mutual prodrugs.

Enzymatic activation of indolequinone-substituted 5-fluorodeoxyuridine prodrugs in hypoxic cells.

Among the various enzymes, reductases that catalyze one-electron reduction are involved in the selective activation of functional compounds or materials in hypoxia, which is one of the well-known pathophysiological characteristics of solid tumors. Enzymatic one-electron reduction has been recognized as a useful reaction that can be applied in the design of tumor hypoxia-targeting drugs. In this report, we characterized the enzymatic reaction of 5-fluorodeoxyuridine (FdUrd) prodrug bearing an indolequinone unit (IQ-FdUrd), which is a substrate of reductases. IQ-FdUrd was activated to release FdUrd under hypoxic conditions after treatment with cytochrome NADPH P450 reductase. We also confirmed that IQ-FdUrd showed selective cytotoxicity in hypoxic tumor cells.

Effect of nucleoside analogue antimetabolites on the structure of PEO-PPO-PEO micelles investigated by SANS.

The effect of three nucleoside analogue antimetabolites (5-fluorouracil, floxuridine, and gemcitabine) on the structure of Pluronic L62 copolymer micelles was investigated using small-angle neutron scattering. These antimetabolites used for cancer chemotherapy have analogous molecular structures but different molecular sizes and aqueous solubilities. It was found that the addition of the three antimetabolites slightly reduced the micellar size and aggregation number, and the micellar anisotropy. The added antimetabolites also changed the internal molecular distribution of the micelles as measured by the scattering length densities, resulting in enhanced hydration of the hydrophobic core region of the micelle. The strength of the effect was found to correlate with the molecular properties of the model drugs, i.e. a larger molecular size and a higher aqueous solubility lead to enhanced hydration of the micellar core.

Doxifluridine-conjugated 2-5A analog shows strong RNase L activation ability and tumor suppressive effect.

RNase L is activated by 2',5'-oligoadenylates (2-5A) at subnanomolar levels to cleave single-stranded RNA. We previously reported the hypothesis that the introduction of an 8-methyladenosine residue at the 2'-terminus of the 2-5A tetramer shifts the 2-5A binding site of RNase L. In this study, we synthesized various 5'-modified 2-5A analogs with 8-methyladenosine at the 2'-terminus. The doxifluridine-conjugated 8-methyladenosine-substituted 2-5A analog was significantly more effective as an activator of RNase L than the parent 5'-monophophorylated 2-5A tetramer and showed a tumor suppressive effect against human cervical cancer cells.

Synergistic Combination Chemotherapy of Camptothecin and Floxuridine through Self-Assembly of Amphiphilic Drug-Drug Conjugate.

Combination chemotherapy has been widely applied in cancer treatment; however, the cocktail administration of combination chemotherapy could cause the nonuniform biodistribution of anticancer agents, thus impairing the therapeutic efficacy. In the present study, to address this concern, we proposed a novel strategy of preparing self-assembled nanoparticles from amphiphilic drug-drug conjugate for synergistic combination chemotherapy. The conjugate was synthesized by two-step esterification of hydrophobic camptothecin (CPT) and hydrophilic floxuridine (FUDR) through a linker compound. Because of its amphiphilic nature, the CPT-FUDR conjugate self-assembled into stable nanoparticles which could simultaneously release fixed dosage of the two drugs in cancer cells. In vitro studies demonstrated synergistic anticancer efficacy of the CPT-FUDR nanoparticles including improved cell apoptosis, varied cell cycle arrest, as well as effective inhibition of cancer cell proliferation.

Self-Assembled Nanoparticles of Amphiphilic Twin Drug from Floxuridine and Bendamustine for Cancer Therapy.

We report here an amphiphilic twin drug strategy directly using small molecular hydrophilic and hydrophobic anticancer drugs to self-assemble into nanoparticles with a high and fixed drug content, which can solve problems of anticancer drug delivery including poor water solubility, low therapeutic indices, and severe side effects. The twin drug has been prepared by the esterification of the hydrophilic anticancer drug floxuridine (FdU) with the hydrophobic anticancer drug bendamustine (BdM). Due to its inherent amphiphilicity, the FdU-BdM twin drug can self-assemble into stable and well-defined nanoparticles. After FdU-BdM twin drug enters into cells, the ester linkage between hydrophilic and hydrophobic drugs is readily cleaved by hydrolysis to release free FdU and BdM. Since both FdU and BdM can kill cancer cells, the FdU-BdM twin drug nanoparticles can overcome the multidrug resistance (MDR) of tumor cells and present an excellent anticancer activity. This strategy can be extended to other hydrophilic and hydrophobic anticancer drugs to synthesize amphiphilic twin drugs which can form nanoparticles to self-deliver drugs for cancer therapy.

Mitochondrial induced and self-monitored intrinsic apoptosis by antitumor theranostic prodrug: in vivo imaging and precise cancer treatment.

Activation of apoptosis, the cell death machinery, in tumor cells by organelle-specific delivery of antitumor theranostic agent is the utmost challenge in cancer therapy. Herein, we developed a highly efficient mitochondria-targeting antitumor theranostic prodrug 7 that contained two molecules of drug 5'-deoxy-5-fluorouridine and an apoptotic marker ethidium for self-monitoring intrinsic (mitochondrial) apoptosis after its activation in tumor cells. Theranostic 7 was activated by endogenously produced mitochondrial-overexpressed H2O2 and released drug 5'-deoxy-5-fluorouridine and apoptotic marker ethidium to the tumor cells. The in vitro and in vivo drug release was monitored by observing the fluorescence changes of ethidium. Theranostic 7 exhibited an enhanced cytotoxicity over commercial 5-fluorouracil (an active drug of 5'-deoxy-5-fluorouridine) leading to intrinsic apoptosis monitored by in situ generated ethidium. Enhanced expression of mitochondria-mediated apoptotic genes (NOXA, PUMA, BID, BAX, and BAK), Cyt C, Caspase-3 and -9, and cell surface death receptors was observed after theranostic 7 activation in tumor cells. In vivo and ex vivo xenografts revealed that theranostic 7 significantly inhibited tumor progression and cured the tumor-bearing mice. Such organelle-specific theranostic strategies have great potential for the early diagnosis and precise treatment of cancer.

Rigid 2',4'-difluororibonucleosides: synthesis, conformational analysis, and incorporation into nascent RNA by HCV polymerase.

We report on the synthesis and conformational properties of 2'-deoxy-2',4'-difluorouridine (2',4'-diF-rU) and cytidine (2',4'-diF-rC) nucleosides. NMR analysis and quantum mechanical calculations show that the strong stereoelectronic effects induced by the two fluorines essentially "lock" the conformation of the sugar in the North region of the pseudorotational cycle. Our studies also demonstrate that NS5B HCV RNA polymerase was able to accommodate 2',4'-diF-rU 5'-triphosphate (2',4'-diF-rUTP) and to link the monophosphate to the RNA primer strand. 2',4'-diF-rUTP inhibited RNA synthesis in dinucleotide-primed reactions, although with relatively high half-maximal inhibitory concentrations (IC50 > 50 µM). 2',4'-diF-rU/C represents rare examples of "locked" ribonucleoside mimics that lack a bicyclic ring structure.

Fluorescent Self-Assembled Complexes Based on Glyco-Functionalized G-Quadruplexes as a Targeted Delivery Platform.

Targeted delivery systems combined with the stimuli-responsive release of drug molecules hold noteworthy promise for precision medicine, enabling treatments with enhanced effectiveness and reduced adverse effects. An ideal drug delivery platform with versatile targeting moieties, the capability of combinational payloads, and simple preparation is highly desirable. Herein, we developed pH-sensitive fluorescent self-assembled complexes (SACs) of a galactose-functionalized G-quadruplex (G4) and a coumarin carboxamidine derivative as a targeted delivery platform through the nanoprecipitation method. These SACs selectively targeted hepatocellular carcinoma (HepG2) cells in fluorescence imaging after a short incubation and exerted specific anticancer effects in an appropriate dose range. Co-delivery of 1 µM prodrug floxuridine oligomers and 16 µg/mL SACs (minimal hemolytic effect) significantly reduced the cytotoxicity of the nucleoside anticancer drug on normal cells (NIH/3T3), kept up to 70% alive after 72-h incubation, and improved anticancer efficacy compared to SACs alone. This strategy can be extended to ratiometric multidrug delivery through self-assembly for targeted combinational therapy.

High-load nanoparticles with a chemotherapeutic SN-38/FdUMP drug cocktail.

[Gd(OH)]2+[(SN-38)0.5(FdUMP)0.5]2- inorganic-organic hybrid nanoparticles (IOH-NPs) with a chemotherapeutic cocktail of ethyl-10-hydroxycamptothecin (SN-38, active form of irinotecan) and 5-fluoro-2'-deoxyuridine-5'-phosphate (FdUMP, active form of 5'-fluoruracil), 40 nm in size, are prepared in water. The IOH-NPs contain a total drug load of 63 wt% with 33 wt% of SN-38 and 30 wt% of FdUMP. Cell-based assays show efficient cellular uptake and promising anti-tumour activity on two pancreatic cancer cell lines of murine origin (KPC, Panc02). Beside the high-load drug cocktail, especially the option to use SN-38, which - although 100- to 1000-times more potent than irinotecan - is usually unsuitable for systemic administration due to poor solubility, low stability, and high toxicity upon non-selective delivery. The [Gd(OH)]2+[(SN-38)0.5(FdUMP)0.5]2- IOH-NPs are a new concept to deliver a drug cocktail with SN-38 and FdUMP directly to the tumour, shielded in a nanoparticle, to reduce side effects.

Esterase-Responsive Floxuridine-Tethered Multifunctional Nanoparticles for Targeted Cancer Therapy.

Floxuridine is a potential clinical anticancer drug for the treatment of various cancers. However, floxuridine typically causes unfavorable side effects due to its very poor tumor selectivity, and, hence, there is a high demand for the development of novel approaches that permit the targeted delivery of floxuridine into cancerous cells. Herein, the design and synthesis of an esterase-responsive multifunctional nanoformulation for the targeted delivery of floxuridine in esterase-overexpressed cancer cells is reported. Photopolymerization of floxuridine-tethered lipoic acid results in the formation of amphiphilic floxuridine-tethered poly(disulfide). Self-assembly of the amphiphilic polymer results in the formation of nanoparticles with floxuridine decorated on the surfaces of the particles. Integration of aptamer DNA for nucleolin onto the surface of the nanoparticle is demonstrated by exploring the base-pairing interaction of floxuridine with adenine. Targeted internalization of the aptamer-decorated nanoparticle into nucleolin-expressed cancer cells is demonstrated. Esterase triggered cleavage of the ester bond connecting floxuridine with the polymer backbone, and the subsequent targeted delivery of floxuridine into cancer cells is also shown. Excellent therapeutic efficacy is observed both in vitro and also in the 3D tumor spheroid model. This noncovalent strategy provides a simple yet effective strategy for the targeted delivery of floxuridine into cancer cells in a less laborious fashion.

Development of a Supermolecular Radionuclide-Drug Conjugate System for Integrated Radiotheranostics for Non-small Cell Lung Cancer.

Radionuclide-drug conjugates (RDCs) designed from small molecule or nanoplatform shows complementary characteristics. We constructed a new RDC system with integrated merits of small molecule and nanoplatform-based RDCs. Erlotinib was labeled with 131I to construct the bulk of RDC (131I-ER). Floxuridine was mixed with 131I-ER to develop a hydrogen bond-driving supermolecular RDC system (131I-ER-Fu NPs). The carrier-free 131I-ER-Fu NPs supermolecule not only demonstrated integrated merits of small molecule and nanoplatform-based RDC, including clear structure definition, stable quality control, prolonged circulation lifetime, enhanced tumor specificity and retention, and rapidly nontarget clearance, but also exhibited low biological toxicity and stronger antitumor effects. In vivo imaging also revealed its application for tumor localization of nonsmall cell lung cancer (NSCLC) and screening of patients suitable for epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy. We considered that 131I-ER-Fu NPs showed potentials as an integrated platform for the radiotheranostics of NSCLC.

Zn2+ selectively stabilizes FdU-substituted DNA through a unique major groove binding motif.

We report, based on semi-empirical calculations, that Zn(2+) binds duplex DNA containing consecutive FdU-dA base pairs in the major groove with distorted trigonal bipyramidal geometry. In this previously uncharacterized binding motif, O4 and F5 on consecutive FdU are axial ligands while three water molecules complete the coordination sphere. NMR spectroscopy confirmed Zn(2+) complexation occurred with maintenance of base pairing while a slight hypsochromic shift in circular dichroism (CD) spectra indicated moderate structural distortion relative to B-form DNA. Zn(2+) complexation inhibited ethidium bromide (EtBr) intercalation and stabilized FdU-substituted duplex DNA (ΔT(m) > 15 °C). Mg(2+) neither inhibited EtBr complexation nor had as strong of a stabilizing effect. DNA sequences that did not contain consecutive FdU were not stabilized by Zn(2+). A lipofectamine preparation of the Zn(2+)-DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn(2+)-DNA complexes for cancer treatment.

Reductive activation of 5-fluorodeoxyuridine prodrug possessing azide methyl group by hypoxic X-irradiation.

We prepared a 5-fluorodeoxyuridine (5-FdUrd) derivative possessing azide methyl group (N(3)-FdUrd) as a novel radiation-activated prodrug. The parent antitumor agent, 5-FdUrd, was released efficiently from N(3)-FdUrd by hypoxic X-irradiation. On the other hand, the activation of N(3)-FdUrd was suppressed upon X-irradiation under aerobic conditions. A biological assay using A549 cells revealed that the cytotoxicity of N(3)-FdUrd was significantly enhanced by hypoxic X-irradiation.

Synthesis and one-electron reduction characteristics of radiation-activated prodrugs possessing two 5-fluorodeoxyuridine units.

Two molecules of an antitumor agent, 5-fluorodeoxyuridine (5-FdUrd), were connected by a 2-oxoalkyl linker (Oxo-linker) at the N(3) position to obtain radiation-activated prodrugs, FdUrd(2) A and FdUrd(2) B. The prodrugs in this study released 5-FdUrd via one-electron reduction initiated by hypoxic X-irradiation. The release of 5-FdUrd from FdUrd(2) A and FdUrd(2) B proceeded more efficiently than that of previous prodrug, Oxo-FdUrd, which possessed one molecule of 5-FdUrd. FdUrd(2) A exhibited increased cytotoxicity against A549 cells when the FdUrd(2) A solution had been irradiated with a large dose of X-rays before administration to the cells. However, we observed no effect on cytotoxicity when the cells were X-irradiated under hypoxic conditions in the presence of FdUrd(2) A because the amount of 5-FdUrd released in the cells seemed to be too low to induce cytotoxic activity.