Gene/Protein Disease Symptom Drug Enzyme Compound
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A general method for isotopic labeling of the purine base moiety of nucleotides and RNA has been developed through biochemical pathway engineering in vitro. A synthetic scheme was designed and implemented utilizing recombinant enzymes from the pentose phosphate and de novo purine synthesis pathways, with regeneration of folate, aspartate, glutamine, ATP, and NADPH cofactors, in a single-pot reaction. Syntheses proceeded quickly and efficiently in comparison to chemical methods with isolated yields up to 66% for 13C-, 15N-enriched ATP and GTP. The scheme is robust and flexible, requiring only serine, NH4+, glucose, and CO2 as stoichiometric precursors in labeled form. Using this approach, U-13C- GTP, U-13C, 15N- GTP, 13C 2,8- ATP, and U-15N- GTP were synthesized on a millimole scale, and the utility of the isotope labeling is illustrated in NMR spectra of HIV-2 transactivation region RNA containing 13C 2,8-adenosine and 15N 1,3,7,9,2-guanosine. Pathway engineering in vitro permits complex synthetic cascades to be effected, expanding the applicability of enzymatic synthesis.
ACS Chem Biol 2008 Aug 15
PMID:Pathway engineered enzymatic de novo purine nucleotide synthesis. 1870 56

Gluconeogenesis is blocked in a strain of Escherichia coli that is deficient in triosephosphate isomerase, but it was restored by the insertion of a plasmid coding for an L-glyceraldehyde 3-phosphate reductase (YghZ). This reductase provides a "bypass" that produces dihydroxyacetone phosphate (DHAP) by the consecutive enzyme-catalyzed reduction of L-glyceraldehyde 3-phosphate ( L-GAP) by NADPH to give L-glycerol 3-phosphate and reoxidation by NAD(+) catalyzed by endogenous L-glycerol 3-phosphate dehydrogenase to give DHAP. The origin of cellular L-GAP remains to be determined.
ACS Chem Biol 2008 Oct 17
PMID:Restoring a metabolic pathway. 1892 48

We address an outstanding issue associated with the biocompatibility of gold nanorods (GNRs), a promising agent for biomedical imaging and theragnostics. GNRs are typically prepared in the presence of cetyltrimethylammonium bromide (CTAB), a cationic surfactant whose rigorous removal is necessary due to its cytotoxicity and membrane-compromising properties. CTAB-stabilized GNRs can be partially purified by treatment with polystyrenesulfonate (PSS), an anionic polyelectrolyte often used as a surrogate peptizing agent, followed by chloroform extraction and ultrafiltration with minimal loss of dispersion stability. However, in vitro cytotoxicity assays of PSS-coated GNRs revealed IC(50) values in the low to submicromolar range, with subsequent studies indicating the source of toxicity to be associated with a persistent PSS-CTAB complex. Further exchange of CTAB-laden PSS with fresh polyelectrolyte greatly improves biocompatibility, to the extent that 85 microg/mL of "CTAB-free" GNRs (the highest level evaluated) has comparable toxicity to a standard phosphate buffer solution. Ironically, PSS is not effective by itself at stabilizing GNRs in CTAB-depleted suspensions: while useful as a detergent for GNR detoxification, it should be replaced by more robust coatings for long-term stability under physiological conditions.
ACS Nano 2008 Dec 23
PMID:Detoxification of gold nanorods by treatment with polystyrenesulfonate. 1920 82

Early detection is a crucial element for the timely diagnosis and successful treatment of all human cancers but is limited by the sensitivity of current imaging methodologies. We have synthesized and studied bioresorbable calcium phosphate nanoparticles (CPNPs) in which molecules of the near-infrared (NIR) emitting fluorophore, indocyanine green (ICG), are embedded. The ICG-CPNPs demonstrate exceptional colloidal and optical characteristics. Suspensions consisting of 16 nm average diameter particles are colloidally stable in physiological solutions (phosphate buffered 0.15 M saline (PBS), pH 7.4) with carboxylate or polyethylene glycol (PEG) surface functionality. ICG-doped CPNPs exhibit significantly greater intensity at the maximum emission wavelength relative to the free constituent fluorophore, consistent with the multiple molecules encapsulated per particle. The quantum efficiency per molecule of the ICG-CPNPs is 200% greater at 0.049 +/- 0.003 over the free fluorophore in PBS. Photostability based on fluorescence half-life of encapsulated ICG in PBS is 500% longer under typical clinical imaging conditions relative to the free dye. PEGylated ICG-CPNPs accumulate in solid, 5 mm diameter xenograft breast adenocarcinoma tumors via enhanced retention and permeability (EPR) within 24 h after systemic tail vein injection in a nude mouse model. Ex situ tissue imaging further verifies the facility of the ICG-CPNPs for deep-tissue imaging with NIR signals detectable from depths up to 3 cm in porcine muscle tissue. Our ex vivo and in vivo experiments verify the promise of the NIR CPNPs for diagnostic imaging in the early detection of solid tumors.
ACS Nano 2008 Oct 28
PMID:Near-infrared emitting fluorophore-doped calcium phosphate nanoparticles for in vivo imaging of human breast cancer. 1920 41

Phosphate sensors were developed by embedding fluorescent reporter proteins (FLIPPi) in polyacrylamide nanoparticles with diameters from 40 to 120 nm. The sensor activity and protein loading efficiency varied according to nanoparticle composition, that is, the total monomer content (% T) and the cross-linker content (% C). Nanoparticles with 28% T and 20% C were considered optimal as a result of relatively high loading efficiency (50.6%) as well as high protein activity (50%). The experimental results prove that the cross-linked polyacrylamide matrix could protect FLIPPi from degradation by soluble proteases to some extent. This nanoparticle embedding method provides a novel promising tool for in vivo metabolite studies. It also demonstrates a universal method for embedding different fragile bioactive elements, such as antibodies, genes, enzymes, and other functional proteins, in nanoparticles for, for example, sensing, biological catalysis, and gene delivery.
ACS Nano 2008 Jan
PMID:Phosphate sensing by fluorescent reporter proteins embedded in polyacrylamide nanoparticles. 1920 43

We show that light-assisted oxidation with hydrogen peroxide effectively and rapidly opens holes in carbon nanohorn walls and, more importantly, creates abundant oxygenated groups such as carboxylic groups at the hole edges. These oxygenated groups reacted with the protein bovine serum albumin. The obtained conjugates were highly dispersed in phosphate-buffered saline and were taken up by cultured mammalian cells via an endocytosis pathway.
ACS Nano 2007 Nov
PMID:Light-assisted oxidation of single-wall carbon nanohorns for abundant creation of oxygenated groups that enable chemical modifications with proteins to enhance biocompatibility. 1920 76

In this article, we report the synthesis of bifunctional Au-Fe(3)O(4) nanoparticles that are formed by chemical bond linkage. Due to the introduction of Au nanoparticles, the resulting bifunctional Au-Fe(3)O(4) nanoparticles can be easily modified with other functional molecules to realize various nanobiotechnological separations and detections. Here, as an example, we demonstrate that as-prepared Au-Fe(3)O(4) nanoparticles can be modified with nitrilotriacetic acid molecules through Au-S interaction and used to separate proteins simply with the assistance of a magnet. Bradford protein assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were performed to examine the validity of the separation procedure, and the phosphate determination method suggested that the as-separated protein maintained catalytic activity. This result shows the efficiency of such a material in protein separation and suggests that its use can be extended to magnetic separation of other biosubstances. Moreover, this synthetic strategy paves the way for facile preparation of diverse bifunctional and even multifunctional nanomaterials.
ACS Nano 2007 Nov
PMID:Bifunctional Au-Fe3O4 nanoparticles for protein separation. 1920 79

In the present study, we report the novel application of polyhydroxylated fullerenes (fullerenols) in cancer drug delivery. The facile synthetic procedure for generating multiple hydroxyl groups on the fullerene cage offers scope for high drug loading in addition to conferring hydrophilicity. Doxorubicin, a first line cancer chemotherapeutic, was conjugated to fullerenols through a carbamate linker, achieving ultrahigh loading efficiency. The drug-fullerenol conjugate was found to be relatively stable in phosphate buffer saline but temporally released the active drug when incubated with tumor cell lysate. The fullerenol-doxorubicin conjugate suppressed the proliferation of cancer cell-lines in vitro through a G2-M cell cycle block, resulting in apoptosis. Furthermore, in an in vivo murine tumor model, fullerenol-doxorubicin exhibited comparable antitumor efficacy as free drug without the systemic toxicity of free doxorubicin. Additionally, we demonstrate that the fullerenol platform can be extended to other chemotherapeutic agents, such as the slightly water-soluble cisplatin, and can emerge as a new paradigm in the management of cancer.
ACS Nano 2009 Sep 22
PMID:Fullerenol-cytotoxic conjugates for cancer chemotherapy. 1968 36

We describe protein- and oligonucleotide-loaded layer-by-layer (LbL)-assembled multilayer films incorporating a hydrolytically degradable polymer for transcutaneous drug or vaccine delivery. Films were constructed based on electrostatic interactions between a cationic poly(beta-amino ester) (denoted Poly-1) with a model protein antigen, ovalbumin (ova), and/or immunostimulatory CpG (cytosine-phosphate diester-guanine-rich) DNA oligonucleotide adjuvant molecules. Linear growth of nanoscale Poly-1/ova bilayers was observed. Dried ova protein-loaded films rapidly deconstructed when rehydrated in saline solutions, releasing ova as nonaggregated/nondegraded protein, suggesting that the structure of biomolecules integrated into these multilayer films is preserved during release. Using confocal fluorescence microscopy and an in vivo murine ear skin model, we demonstrated delivery of ova from LbL films into barrier-disrupted skin, uptake of the protein by skin-resident antigen-presenting cells (Langerhans cells), and transport of the antigen to the skin-draining lymph nodes. Dual incorporation of ova and CpG oligonucleotides into the nanolayers of LbL films enabled dual release of the antigen and adjuvant with distinct kinetics for each component; ova was rapidly released, while CpG was released in a relatively sustained manner. Applied as skin patches, these films delivered ova and CpG to Langerhans cells in the skin. To our knowledge, this is the first demonstration of LbL films applied for the delivery of biomolecules into skin. This approach provides a new route for storage of vaccines and other immunotherapeutics in a solid-state thin film for subsequent delivery into the immunologically rich milieu of the skin.
ACS Nano 2009 Nov 24
PMID:Layer-by-layer-assembled multilayer films for transcutaneous drug and vaccine delivery. 1992 31

Sensitive methods to probe the activity of enzymes are important for clinical assays and for elucidating the role of these proteins in complex biochemical networks. This paper describes a semi-synthetic ion channel platform for detecting the activity of two different classes of enzymes with high sensitivity. In the first case, this method uses single ion channel conductance measurements to follow the enzyme-catalyzed hydrolysis of a phosphate group attached to the C-terminus of gramicidin A (gA, an ion channel-forming peptide) in the presence of alkaline phosphatase (AP). Enzymatic hydrolysis of this phosphate group removes negative charges from the entrance of the gA pore, resulting in a product with measurably reduced single ion channel conductance compared to the original gA-phosphate substrate. This technique employs a standard, commercial bilayer setup and takes advantage of the catalytic turnover of enzymes and the amplification characteristics of ion flux through individual gA pores to detect picomolar concentrations of active AP in solution. Furthermore, this technique makes it possible to study the kinetics of an enzyme and provides an estimate for the observed rate constant (k(cat)) and the Michaelis constant (K(M)) by following the conversion of the gA-phosphate substrate to product over time in the presence of different concentrations of AP. In the second case, modification of gA with a substrate for proteolytic cleavage by anthrax lethal factor (LF) afforded a sensitive method for detection of LF activity, illustrating the utility of ion channel-based sensing for detection of a potential biowarfare agent. This ion channel-based platform represents a powerful, novel approach to monitor the activity of femtomoles to picomoles of two different classes of enzymes in solution. Furthermore, this platform has the potential for realizing miniaturized, cost-effective bioanalytical assays that complement currently established assays.
ACS Nano 2009 Nov 24
PMID:A semi-synthetic ion channel platform for detection of phosphatase and protease activity. 1986 Mar 82


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