DNA Delivery Systems Based on Peptide-Mimicking Cationic Lipids-The Effect of the Co-Lipid on the Structure and DNA Binding Capacity.

In continuation of previous work, we present a new promising DNA carrier, OO4, a highly effective peptide-mimicking lysine-based cationic lipid. The structural characteristics of the polynucleotide carrier system OO4 mixed with the commonly used co-lipid DOPE and the saturated phospholipid DPPE have been studied in two-dimensional and three-dimensional model systems to understand their influence on the physical-chemical properties. The phase behavior of pure OO4 and its mixtures with DOPE and DPPE was studied at the air-water interface using a Langmuir film balance combined with infrared reflection-absorption spectroscopy. In bulk, the self-assembling structures in the presence and absence of DNA were determined by small-angle and wide-angle X-ray scattering. The amount of adsorbed DNA to cationic lipid bilayers was measured using a quartz crystal microbalance. The choice of the co-lipid has an enormous influence on the structure and capability of binding DNA. DOPE promotes the formation of nonlamellar lipoplexes (cubic and hexagonal structures), whereas DPPE promotes the formation of lamellar lipoplexes. The correlation of the observed structures with the transfection efficiency and serum stability indicates that OO4/DOPE 1:3 lipoplexes with a DNA-containing cubic phase encapsulated in multilamellar structures seem to be most promising.

A radical containing injectable in-situ-oleogel and emulgel for prolonged in-vivo oxygen measurements with CW EPR.

Molecular oxygen, reactive oxygen species and free radicals derived from oxygen play important roles in a broad spectrum of physiological and pathological processes. The quantitative measurement of molecular oxygen in tissues by electron paramagnetic resonance (EPR) has great potential for understanding and diagnosing a number of diseases, and for developing and guiding therapies. This requires improvements in the free radical probe systems that sense and report molecular oxygen levels in vivo. We report on the encapsulation of existing free radical probes in lipophilic gel implants: an in-situ-oleogel and an emulgel, based only on well-known, safe excipients for the incorporation of lipophilic and hydrophilic radicals, respectively. The EPR signals of encapsulated radicals were not altered compared to dissolved radicals. The high solubility of oxygen in lipophilic solvents enhanced oxygen sensitivity. The gels extended the lifetime of the radicals in tissues from tens of minutes to many days, simplifying studies with extended series of measurements. The encapsulated radicals showed a good in vivo response to changes in oxygen supply and seem to circumvent concerns from toxicity of the radical probes. These gels simplify the development of new oxygen-sensitive free radical probes for EPR oximetry by making their in vivo stability, persistence and toxicity a function of the encapsulating gel and not a set of additional requirements for the free radical probe.

EPR Oximetry Sensor-Developing a TAM Derivative for In Vivo Studies.

Oxygenation is one of the most important physiological parameters of biological systems. Low oxygen concentration (hypoxia) is associated with various pathophysiological processes in different organs. Hypoxia is of special importance in tumor therapy, causing poor response to treatment. Triaryl methyl (TAM) derivative radicals are commonly used in electron paramagnetic resonance (EPR) as sensors for quantitative spatial tissue oxygen mapping. They are also known as magnetic resonance imaging (MRI) contrast agents and fluorescence imaging compounds. We report the properties of the TAM radical tris(2,3,5,6-tetrachloro-4-carboxy-phenyl)methyl, (PTMTC), a potential multimodal (EPR/fluorescence) marker. PTMTC was spectrally analyzed using EPR and characterized by estimation of its sensitivity to the oxygen in liquid environment suitable for intravenous injection (1 mM PBS, pH = 7.4). Further, fluorescent emission of the radical was measured using the same solvent and its quantum yield was estimated. An in vitro cytotoxicity examination was conducted in two cancer cell lines, HT-29 (colorectal adenocarcinoma) and FaDu (squamous cell carcinoma) and followed by uptake studies. The stability of the radical in different solutions (PBS pH = 7.4, cell media used for HT-29 and FaDu cells culturing and cytotoxicity procedure, full rat blood and blood plasma) was determined. Finally, a primary toxicity test of PTMTC was carried out in mice. Results of spectral studies confirmed the multimodal properties of PTMTC. PTMTC was demonstrated to be not absorbed by cancer cells and did not interfere with luciferin-luciferase based assays. Also in vitro and in vivo tests showed that it was non-toxic and can be freely administrated till doses of 250 mg/kg BW via both i.v. and i.p. injections. This work illustrated that PTMTC is a perfect candidate for multimodal (EPR/fluorescence) contrast agent in preclinical studies.