Nucleic Acids as a Nature‐Inspired Scaffold for Macromolecular Prodrugs of Nucleoside Analogues

Abstract Macromolecular prodrugs (MP) built on the natural phosphodiester and deoxyribose backbone are developed using marketed antiviral nucleoside analogues. These MP are synthesized using automated synthesis, have defined molecular composition, and have a natural mechanism for drug release. These unique attributes, coupled to the efficient cell entry and potent antiviral effects, position the prodrugs scaffolded on nucleic acids favorably for translational studies.

General procedure for DMT protection of 3, 4 and 9 compounds: Starting material (1 equiv.) was dissolved in dry pyridine and DMT-Cl (1.2 equiv.) was added and reaction was stirred overnight under argon condition. Pyridine was evaporated under high vacuum and ethyl acetate was added to oily paste then, washed with sat. NaHCO 3 and brine and dried over sodium sulfate. The organic solvent was removed by rotary evaporator and the compound was purified by column chromatography (MeOH/ DCM with 0.5% triethyl amine). General procedure for phosphoramidite synthesis for 5, 6: Anhydrous dichloromethane was added to the DMT alcohol (3 or 4, 1 equiv) followed by the addition of diisopropyl ammonium tetrazolide (0.7 equiv). Subsequently, 2-cyanoethyl N,N,N',N'tetraisopropylphosphordiamidite (1.3 equiv) was added and allowed to stir at room temperature for 3 hr. The reaction was diluted with dichloromethane and poured in saturated sodium bicarbonate aqueous solution. The product was extracted and dried over dry sodium sulfate. Compound was purified by column chromatography.

Synthesis of oligonucleotides:
Standard protocol of phosphoramidite chemistry was used for synthesis of oligonucleotides in which modified and standard monomers were dissolved (100 mM solution) in anhydrous dichloromethane and anhydrous acetonitrile respectively. Each monomer coupling was performed for 2 min extended time in case of modified monomers. Deprotection and oligonucleotides cleavage from solid support were carried out by 50 mM potassium carbonate in dry methanol for 4 hours at room temperature. Oligonucleotides were purified by Glen-Pak™ DNA cartridge. Purity was analyzed by reverse phase HPLC. Cy5 dye was conjugated with maleimide chemistry and conjugates were purified by reverse phase HPLC. Oligonucleotides were analyzed by liquid chromatography-mass spectrometry (the mass of ON-11 was determined by MALDI-TOF).  Cy5III III III III IIC-3

Determination of infection by cytopathic effect and cytotoxicity of therapeutic nucleic acids using a CellTiter-Glo ® (CTG) luminescent cell viability assay
To determine the antiviral and cytotoxic effects of therapeutic nucleic acids, 6,000 Vero E6 cells per well were seeded in 96-well flat bottom plate in 100 µl medium. The next day, medium was removed and 70 µl fresh medium was added. TNA and controls were diluted in PBS twofold, starting with a concentration of 200 mg/L. 10 µl were added to cells and incubated for 2 h in CO 2 incubator at 37°C. Cells were then infected with an MOI of 0.02 of HSV-2 eGFP (333) or 20 µl fresh medium was added. 72 h later the ATP levels were quantitated. Therefore, the plates were exposed to room temperature. After 15 min 50 µl of buffer-substrate-mix of CellTiter-Glo ® (CTG) luminescent cell viability assay was added to each well. After 10 min incubation in the dark, the solution was transferred into white microtiter plate and ATP-dependent luminescence was measured using Orion II microplate luminometer (Software Simplicity, 0.1 s measurement). Infection rates were determined by subtracting sample values from untreated control and untreated control was set to 100%.
Determination of infection by cytopathic effect and cytotoxicity of compounds using a Colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)based cell viability assay The inhibitory effect of therapeutic nucleic acids against clinical isolates of HSV-1 and HSV-2 was determined by incubating 6,000 Vero E6 cells seeded the day before in 96-well flat bottom plate in 100 µl medium. The supernatant was removed, 70 µl fresh medium and 10 µl of five-fold diluted therapeutic nucleic acids and respective controls starting with a concentration of 200 mg/l was added. 2 h later cells were infected with 20 µl of clinical isolates with an MOI of 0.02. 72 h afterwards, bright field images were taken with a Leica MC120 HD colour camera connected to a microscope (Europa Promotion Leica DM IL LED, Leica microsystems) with a 10x objective. Subsequently, supernatant was removed and 100 µl of 1 to 10 diluted MTT (5 mg/ml) solution was added. After 2.5 h at 37°C supernatant was discarded and formazan crystals were dissolved in 100 µl 1:1 DMSO-EtOH solution. Absorption was measured at 450 nm and baseline was corrected at 650 nm using a Vmax kinetic microplate reader. To determine infection rates, sample values were subtracted from untreated control and untreated control set to 100%. The same was done for therapeutic nucleic acids conjugated to gold nanoparticles. Gold nanoparticles were dissolved five-fold starting with a concentration of 20 mg/l. Infection was carried out with 20 µl of HSV-2 eGFP (333) and an MOI of 0.02. The determination of infection by cytopathic effect was done as indicated above.
To assess the extracellular stability of the therapeutic nucleic acids 12,000 Vero E6 cells were seeded in 96-well flat bottom plate in 100 µl medium. The next day, medium was removed, cells were carefully washed with PBS and 70 µl X-VIVO TM 15 Serum-free Hematopoietic Cell Medium (Lonza) supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, 1 mM sodium pyruvate and 1x non-essential amino acids (Invitrogen, Glostrup, Denmark) was added. 72 h post infection in X-VIVO TM medium, following procedure is equal to the one indicated before using therapeutic nucleic acids, an infection with clinical isolates and determination of infection by cytopathic effect using MTT.

Determination of uptake of therapeutic nucleic acids and inhibitory effect against clinical isolates in human cell lines
To investigate the uptake of therapeutic nucleic acids, 40,000 lung epithelial carcinoma cells (A549) and human foreskin fibroblast (HFF) cells were seeded in 8-well Ibidi slide in 150 µl. The next day, supernatant was discarded and 120 µl fresh medium was added. Cy5-labelled therapeutic nucleic acids were diluted 5 fold, starting with a concentration of 200 mg/l, incubated for 2 h at 37°C and subsequently, cells were infected with 15 µl with HSV-1 isolate from broncho-alveolar lavage (A549) or HSV-2 isolate from vaginal swab (HFF) with an MOI 0.02. After 72 h hours at 37°C, supernatant was discarded and cells were fixed with 4% paraformaldehyde for 10 min at 4°C. All staining steps were carried out at room temperature and between every step, cells were washed twice with PBS. Firstly, a blocking solution containing PBS with 10% (v/v) bovine serum albumin (BSA) and 5% (v/v) FCS was added for 30 min. To visualize the infection with HSV, cells infected with HSV-1 were incubated with a primary antibody against gE (sc-58153; Santa Cruz Biotechnology, Inc.) diluted 1 to 500 (v/v) and cells infected with HSV-2 were incubated with a primary antibody against gD (ab6507; abcam ® ) diluted    Figure S7. Infectivity of the HSV-2 in Vero E6 cells in the presence of idoxuridine or the Au nanoparticles equipped with TNA. Cytopathic effect and therefore infection was determined using the colorimetric MTT cell viability assay. n=2 in triplicates, SD.