Anticancer Drug-DNA Interactions Measured Using a Photoinduced Electron-Transfer Mechanism Based on Luminescent Quantum Dots

A sensing system based on the photoinduced electron transfer of quantum dots (QDs) was designed to measure the interaction of anticancer drug and DNA, taking mitoxantrone (MTX) as a model drug. MTX adsorbed on the surface of QDs can quench the photoluminescence (PL) of QDs through the photoinduced electron-transfer process; and then the addition of DNA will bring the restoration of QDs PL intensity, as DNA can bind with MTX and remove it from QDs. Sensitive detection of MTX with the detection limit of 10 nmol L-1 and a linear detection range from 10 nmol L-1 to 4.5 mu mol L-1 was achieved. The dependence of PL intensity on DNA amount was successfully utilized to investigate the interactions between MTX and DNA. Both the binding constants and the sizes of binding site of MTX-DNA interactions were calculated based on the equations deduced for the PL recovery process. The binding constant obtained in our experiment was generally consistent with previous reports. The sensitive and speedy detection of MTX as well as the avoidance of modification or immobilization process made this system suitable and promising in the drug-DNA interaction studies.

Daunomycin binding to detergent micelles: A model system for evaluating the hydrophobic contribution to drug-DNA interactions

The interaction of daunomycin with sodium dodecyl sulfate and Triton X-100 micelles was investigated as a model for the hydrophobic contribution to the free energy of DNA intercalation reactions. Measurements of visible absorbance, fluorescence lifetime, steady-state fluorescence emission intensity, and fluorescence anisotropy indicate that the anthraquinone ring partitions into the hydrophobic micelle interior. Fluorescence quenching experiments using both steady-state and lifetime measurements demonstrate reduced accessibility of daunomycin in sodium dodecyl sulfate micelles to the anionic quencher iodide and to the neutral quencher acrylamide. Quenching of daunomycin fluorescence by iodide in Triton X-100 micelles was similar to that seen with free daunomycin. Studies of the energetics of the interaction of daunomycin with micelles by fluorescence and absorbance titration methods and by isothermal titration calorimetry in the presence of excess micelles revealed that association with sodium dodecyl sulfate and Triton X-100 micelles is driven by a large negative enthalpy. Association of the drug with both types of micelles also has a favorable entropic contribution, which is larger in magnitude for Triton X-100 micelles than for sodium dodecyl sulfate micelles.

Polymeric nanoparticles : from microporosity and drug release kinetics to cellular interactions

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Stochiometrically governed molecular interactions in drug: Poloxamer solid dispersions

This study probes the molecular interactions between model drugs and poloxamers that facilitate dissolution rate improvements using solid dispersions. Ibuprofen and ketoprofen solid dispersions were prepared at different mole ratios using poloxamers 407 and 188. The carbonyl stretching vibration of the ibuprofen dimer shifted to higher wavenumber in the infrared spectra of 2:1 drug:carrier mole ratio solid dispersions, indicating disruption of the ibuprofen dimer concomitant with hydrogen bond formation between the drug and carrier. Solid dispersions with mole ratios >2:1 drug:carrier (up to 29:1) showed both ibuprofen hydrogen-bonded to the poloxamer, and excess drug present as dimers. X-ray diffraction studies confirmed these findings with no evidence of crystalline drug in 2:1 mole ratio systems whereas higher drug loadings retained crystalline ibuprofen. Similar results were found with ketoprofen-poloxamer solid dispersions. Thermal analysis of ibuprofen-poloxamer 407 solid dispersions and their resultant phase diagram suggested solid solutions and a eutectic system were formed, depending on drug loading. Dissolution studies showed fastest release from the solid solutions; dissolution rates from solid solutions were 12-fold greater than the dissolution of ibuprofen powder whereas the eutectic system gave a 6-fold improvement over the powder. When designing solid dispersions to improve the delivery of poorly-water soluble drugs, the nature of drug:carrier interactions, which are governed by the stochiometry of the composition, can affect the dissolution rate improvement.

Noncovalent interactions of drugs with immune receptors may mediate drug-induced hypersensitivity reactions

Drug-induced hypersensitivity reactions are instructive examples of immune reactions against low molecular weight compounds. Classically, such reactions have been explained by the hapten concept, according to which the small antigen covalently modifies an endogenous protein; recent studies show strong associations of several HLA molecules with hypersensitivity. In recent years, however, evidence has become stronger that not all drugs need to bind covalently to the major histocompatibility complex (MHC)-peptide complex in order to trigger an immune response. Rather, some drugs may bind reversibly to the MHC or possibly to the T-cell receptor (TCR), eliciting immune reactions akin to the pharmacological activation of other receptors. While the exact mechanism is still a matter of debate, noncovalent drug presentation clearly leads to the activation of drug-specific T cells. In some patients with hypersensitivity, such a response may occur within hours of even the first exposure to the drug. Thus, the reaction to the drug may not be the result of a classical, primary response but rather be mediated by existing, preactivated T cells that display cross-reactivity for the drug and have additional (peptide) specificity as well. In this way, certain drugs may circumvent the checkpoints for immune activation imposed by the classical antigen processing and presentation mechanisms, which may help to explain the idiosyncratic nature of many drug hypersensitivity reactions.

Will diverse Tat interactions lead to novel antiretroviral drug targets?

More than fifteen years following the description of Tat as a critical HIV gene expression regulatory protein, additional roles for Tat in HIV replication have been described, including reverse transcription. Tat achieves function through direct interaction with viral proteins, including reverse transcriptase, and numerous cellular proteins including cyclin T1, RNA polymerase 11, protein kinase R (PKR), p300/CBP, and P/CAF. Despite our advanced knowledge of how Tat operates, this has not yet resulted in the discovery of effective agents capable of targeting various Tat functions. Nevertheless, Tat remains an attractive, virus-specific molecule and detailed understanding of specific protein interaction holds promise for future drug discovery.

Drug/Water interactions in hydrogel matrices

The primary aim of this research has been the investigation of the role of water structuring effects in the widely different extents of irritancy displayed by certain antibiotics. The compounds involved were members of the Lincomycin group of antibiotics. The aqueous solution behaviour of these co~pounds was studied using techniques such as vapour pressure osmometry end differential scanning calorimetry (D.S.C.). The effects of the antibiotics on water structure in hydrogel membrane preparations In which the equilibrium water content (E.W.C.) and constituent amounts of freezing and non-freezing water ware varied were also investigated using D.S.C. The permeability of water swollen hydrogel preparations to aqueous antibiotic solutions as well as other solutes were studied. A series of hydrogel preparations into which the antibiotics had been incorporated during polymerisation were developed and used in studies of the effects of the antibiotics end their water structure modifications on the permeation of a range of solutes.

Applications of functional nucleic acids in imaging, sensing and drug delivery and investigations of DNAzyme-metal interactions

Functional nucleic acids (FNA), including nucleic acids catalysts (ribozymes and DNAzymes) and ligands (aptamers), have been discovered in nature or isolated in a laboratory through a process called in vitro selection. They are nucleic acids with functions similar to protein enzymes or antibodies. They have been developed into sensors with high sensitivity and selectivity; it is realized by converting the reaction catalyzed by a DNAzyme/ribozyme or the binding event of an aptamer to a fluorescent, colorimetric or electrochemical signal. While a number of studies have been reported for in vitro sensing using DNAzymes or aptamers, there are few reports on in vivo sensing or imaging. MRI is a non-invasive imaging technique; smart MRI contrast agents were synthesized for molecular imaging purposes. However, their rational design remains a challenge due to the difficulty to predict molecular interactions. Chapter 2 focuses on rational design of smart T1-weighted MRI contrast agents with high specificity based on DNAzymes and aptamers. It was realized by changing the molecular weight of the gadolinium conjugated DNA strand with the analytes, which lead to analyte-specific water proton relaxation responses and contrast changes on an MRI image. The designs are general; the high selectivity of FNA was retained. Most FNA-based fluorescent sensors require covalent labeling of fluorophore/quencher to FNAs, which incurrs extra expenses and could interfere the function of FNAs. Chapter 3 describes a new sensor design avoiding the covalent labeling of fluorophore and quencher. The fluorescence of malachite green (MG) was regulated by the presence of adenosine. Conjugate of aptamers of MG and adenosine and a bridge strand were annealed in a solution containing MG. The MG aptamer did not bind MG because of its hybridization to the bridge strand, resulting in low fluorescence signal of MG. The hybridization was weakened in the presence of adenosine, leading to the binding of MG to its aptamer and a fluorescence increase. The sensor has comparable detection limit (20 micromolar) and specificity to its labeled derivatives. Enzymatic activity of most DNAzymes requires metal cations. The research on the metal-DNAzyme interaction is of interest and challenge to scientists because of the lack of structural information. Chapters 4 presents the research on the characterization of the interaction between a Cu2+-dependent DNAzyme and Cu2+. Electron paramagnetic resonance (EPR) and UV-Vis spectroscopy were used to probe the binding of Cu2+ to the DNAzyme; circular dichroism was used to probe the conformational change of the DNAzyme induced by Cu2+. It was proposed that the conformational change by the Cu2+ binding is important for the activity of the DNAzyme. Chapter 5 reports the dependence of the activity of 8-17 DNAzyme on the presence of both Pb2+ and other metal cations including Zn2+, Cd2+ and Mg2+. It was discovered that presence of those metal cations can be cooperative or inhibitive to 8-17 activity. It is hypothesized that the 8-17 DNAzyme had multiple binding sites for metal cations based on the results. Cisplatin is effective killing tumor cells, but with significant side effects, which can be minimized by its targeted delivery. Chapter 6 focuses on the effort to functionalize liposomes encapsulating cisplatin by an aptamer that selectively bind nucleolin, an overexpressed protein by breast cancer cells. The study proved the selective cytotoxicity to breast cancer cells of the aptamer-functionalized liposome.