111In- and IRDye800CW-Labeled PLA-PEG Nanoparticle for Imaging Prostate-Specific Membrane Antigen-Expressing Tissues.

Targeted delivery of drug-encapsulated nanoparticles is a promising new approach to safe and effective therapeutics for cancer. Here we investigate the pharmacokinetics and biodistribution of a prostate-specific membrane antigen (PSMA)-targeted nanoparticle based on a poly(lactic acid)-polyethylene glycol copolymer by utilizing single photon emission computed tomography (SPECT) and fluorescence imaging of a low-molecular-weight, PSMA-targeting moiety attached to the surface and oriented toward the outside environment. Tissue biodistribution of the radioactive, PSMA-targeted nanoparticles in mice containing PSMA(+) PC3 PIP and PSMA(-) PC3 flu (control) tumors demonstrated similar accumulation compared to the untargeted particles within all tissues except for the tumor and liver by 96 h postinjection. For PSMA(+) PC3 PIP tumor, the targeted nanoparticle demonstrated retention of 6.58% injected dose (ID)/g at 48 h and remained nearly at that level out to 96 h, whereas the untargeted nanoparticle showed a 48 h retention of 8.17% ID/g followed by a significant clearance to 2.37% ID/g at 96 h (P < 0.02). On the other hand, for control tumor, both targeted and untargeted particles displayed similar 48 h retentions and rates of clearance over 96 h. Ex vivo microscopic analysis with near-infrared versions of the nanoparticles indicated retention within PSMA(+) tumor epithelial cells as well as tumor-associated macrophages for targeted particles and primarily macrophage-associated uptake for the untargeted particles. Retention in control tumor was primarily associated with tumor vasculature and macrophages. The data demonstrate the utility of radioimaging to assess nanoparticle biodistribution and suggest that active targeting has a modest positive effect on tumor localization of PSMA-targeted PLA-PEG nanoparticles that have been derivatized for imaging.

Synthesis and properties of DNA containing cyclonucleosides.

Here, we present efficient syntheses of the R and S diastereomers of 8,5'-cyclo-2'-deoxyadenosine and 6,5'-cyclo-2'-deoxyuridine. We incorporated these interesting nucleosides into DNA to study how the cyclo linkage affects the stability of duplex formation.

Preclinical development and clinical translation of a PSMA-targeted docetaxel nanoparticle with a differentiated pharmacological profile.

We describe the development and clinical translation of a targeted polymeric nanoparticle (TNP) containing the chemotherapeutic docetaxel (DTXL) for the treatment of patients with solid tumors. DTXL-TNP is targeted to prostate-specific membrane antigen, a clinically validated tumor antigen expressed on prostate cancer cells and on the neovasculature of most nonprostate solid tumors. DTXL-TNP was developed from a combinatorial library of more than 100 TNP formulations varying with respect to particle size, targeting ligand density, surface hydrophilicity, drug loading, and drug release properties. Pharmacokinetic and tissue distribution studies in rats showed that the NPs had a blood circulation half-life of about 20 hours and minimal liver accumulation. In tumor-bearing mice, DTXL-TNP exhibited markedly enhanced tumor accumulation at 12 hours and prolonged tumor growth suppression compared to a solvent-based DTXL formulation (sb-DTXL). In tumor-bearing mice, rats, and nonhuman primates, DTXL-TNP displayed pharmacokinetic characteristics consistent with prolonged circulation of NPs in the vascular compartment and controlled release of DTXL, with total DTXL plasma concentrations remaining at least 100-fold higher than sb-DTXL for more than 24 hours. Finally, initial clinical data in patients with advanced solid tumors indicated that DTXL-TNP displays a pharmacological profile differentiated from sb-DTXL, including pharmacokinetics characteristics consistent with preclinical data and cases of tumor shrinkage at doses below the sb-DTXL dose typically used in the clinic.

Enzymatic primer-extension with glycerol-nucleoside triphosphates on DNA templates.

BACKGROUND: Glycerol nucleic acid (GNA) has an acyclic phosphoglycerol backbone repeat-unit, but forms stable duplexes based on Watson-Crick base-pairing. Because of its structural simplicity, GNA is of particular interest with respect to the possibility of evolving functional polymers by in vitro selection. Template-dependent GNA synthesis is essential to any GNA-based selection system. PRINCIPAL FINDINGS: In this study, we investigated the ability of various DNA polymerases to use glycerol-nucleoside triphosphates (gNTPs) as substrates for GNA synthesis on DNA templates. Therminator DNA polymerase catalyzes quantitative primer-extension by the incorporation of two glyceronucleotides, with much less efficient extension up to five glyceronucleotides. Steady-state kinetic experiments suggested that GNA synthesis by Therminator was affected by both decreased catalytic rates and weakened substrate binding, especially for pyrimidines. In an attempt to improve pyrimidine incorporation by providing additional stacking interactions, we synthesized two new gNTP analogs with 5-propynyl substituted pyrimidine nucleobases. This led to more efficient incorporation of gC, but not gT. CONCLUSIONS: We suggest that directed evolution of Therminator might lead to mutants with improved substrate binding and catalytic efficiency.

Glycerol nucleoside triphosphates: synthesis and polymerase substrate activities.

[Structure: see text] The synthesis of (S)-glycerol nucleoside triphosphates (gNTPs) and the analysis of their substrate activities for enzymatic polymerization is described. NTPs with simplified carbohydrate backbones such as the tNTPs (alpha-L-threose-NTPs) are polymerase substrates and offer the potential to create non-natural aptamer sequences with simplified backbones through enzymatic means. The acyclic (S)-GNA was modeled after the shortened alpha-threofuranosyl backbone. Here we describe the synthesis of (S)-glycerol NTPs and initial enzymatic testing of this further simplified nucleic acid backbone.

High fidelity TNA synthesis by Therminator polymerase.

Therminator DNA polymerase is an efficient DNA-dependent TNA polymerase capable of polymerizing TNA oligomers of at least 80 nt in length. In order for Therminator to be useful for the in vitro selection of functional TNA sequences, its TNA synthesis fidelity must be high enough to preserve successful sequences. We used sequencing to examine the fidelity of Therminator-catalyzed TNA synthesis at different temperatures, incubation times, tNTP ratios and primer/template combinations. TNA synthesis by Therminator exhibits high fidelity under optimal conditions; the observed fidelity is sufficient to allow in vitro selection with TNA libraries of at least 200 nt in length.

Kinetic analysis of an efficient DNA-dependent TNA polymerase.

alpha-l-Threofuranosyl nucleoside triphosphates (tNTPs) are tetrafuranose nucleoside derivatives and potential progenitors of present-day beta-d-2'-deoxyribofuranosyl nucleoside triphosphates (dNTPs). Therminator DNA polymerase, a variant of the 9 degrees N DNA polymerase, is an efficient DNA-directed threosyl nucleic acid (TNA) polymerase. Here we report a detailed kinetic comparison of Therminator-catalyzed TNA and DNA syntheses. We examined the rate of single-nucleotide incorporation for all four tNTPs and dNTPs from a DNA primer-template complex and carried out parallel experiments with a chimeric DNA-TNA primer-DNA template containing five TNA residues at the primer 3'-terminus. Remarkably, no drop in the rate of TNA incorporation was observed in comparing the DNA-TNA primer to the all-DNA primer, suggesting that few primer-enzyme contacts are lost with a TNA primer. Moreover, comparison of the catalytic efficiency of TNA synthesis relative to DNA synthesis at the downstream positions reveals a difference of no greater than 5-fold in favor of the natural DNA substrate. This disparity becomes negligible when the TNA synthesis reaction mixture is supplemented with 1.25 mM MnCl(2). These results indicate that Therminator DNA polymerase can recognize both a TNA primer and tNTP substrates and is an effective catalyst of TNA polymerization despite changes in the geometry of the reactants.

Synthesis of alpha-L-threofuranosyl nucleoside triphosphates (tNTPs).

[structure: see text] The alpha-l-threofuranosyl nucleoside triphosphates of T, G, and D (tTTP, tGTP, and tDTP) were synthesized from the described 2'-O-DMT-protected derivatives using the Eckstein method, while the corresponding C derivative (tCTP) was prepared from the 2'-O-acetyl derivative. The prepared alpha-l-threofuranosyl nucleoside triphosphates, despite being one carbon shorter than the native 2'-deoxyfuranosyl nucleoside triphosphates, are effective substrates for selected DNA polymerases.

An in vitro selection system for TNA.

(3'-2')-alpha-l-Threose nucleic acid (TNA) is an unnatural polymer that possesses the rare ability to base-pair with RNA, DNA, and itself. This feature, coupled with its chemical simplicity, makes TNA of interest as a possible progenitor of RNA during the early history of life. To evaluate the functional potential of TNA, we have developed a system for the in vitro selection of TNA. We identified the Therminator DNA polymerase as a remarkably efficient DNA-dependent TNA polymerase capable of polymerizing more than 50 tNTPs. We have also developed a method of covalently linking a DNA template to the TNA strand that it encodes, thus obviating the need for a TNA-dependent DNA polymerase during cycles of selection.