Relating tissue/organ energy expenditure to metabolic fluxes in mouse and human: experimental data integrated with mathematical modeling.

Mouse models of human diseases are used to study the metabolic and physiological processes leading to altered whole-body energy expenditure (EE), which is the sum of EE of all body organs and tissues. Isotopic techniques, arterio-venous difference of substrates, oxygen, and blood flow measurements can provide essential information to quantify tissue/organ EE and substrate oxidation. To complement and integrate experimental data, quantitative mathematical model analyses have been applied in the design of experiments and evaluation of metabolic fluxes. In this study, a method is presented to quantify the energy expenditure of the main mouse organs using metabolic flux measurements. The metabolic fluxes and substrate utilization of the main metabolic pathways of energy metabolism in the mouse tissue/organ systems and the whole body are quantified using a mathematical model based on mass and energy balances. The model is composed of six organ/tissue compartments: brain, heart, liver, gastrointestinal tract, muscle, and adipose tissue. Each tissue/organ is described with a distinct system of metabolic reactions. This model quantifies metabolic and energetic characteristics of mice under overnight fasting conditions. The steady-state mass balances of metabolites and energy balances of carbohydrate and fat are integrated with available experimental data to calculate metabolic fluxes, substrate utilization, and oxygen consumption in each tissue/organ. The model serves as a paradigm for designing experiments with the minimal reliable measurements necessary to quantify tissue/organs fluxes and to quantify the contributions of tissue/organ EE to whole-body EE that cannot be easily determined currently.

Design and evaluation of new pH-sensitive amphiphilic cationic lipids for siRNA delivery.

Synthetic small interfering RNA (siRNA) has become the basis of a new generation of gene-silencing cancer therapeutics. However, successful implementation of this novel therapy relies on the ability to effectively deliver siRNA into target cells and to prevent degradation of siRNA in lysosomes after endocytosis. In this study, our goal was to design and optimize new amphiphilic cationic lipid carriers that exhibit selective pH-sensitive endosomal membrane disruptive capabilities to allow for the efficient release of their siRNA payload into the cytosol. The pH sensitive siRNA carriers consist of three domains (cationic head, hydrophobic tail, amino acid-based linker). A library of eight lipid carriers were synthesized using solid phase chemistry, and then studied to determine the role of (1) the number of protonable amines and overall pKa of the cationic head group, (2) the degree of unsaturation of the hydrophobic tail, and (3) the presence of histidine residues in the amino acid linker for transfection and silencing efficacy. In vitro screening evaluation of the new carriers demonstrated at least 80% knockdown of a GFP reporter in CHO cells after 72h. The carriers ECO and ECLn performed the best in a luciferase knockdown study in HT29 human colon cancer cells, which were found to be more difficult to transfect. They significantly reduced expression of this reporter to 22.7±3.31% and 23.5±5.11% after 72h post-transfection, better than Lipofectamine RNAiMax. Both ECO and ECLn carriers caused minimal cytotoxicity, preserving relative cell viabilities at 87.3±2.72% and 88.9±6.84%, respectively. A series of hemolysis assays at various pHs revealed that increasing the number of amines in the protonable head group, and removing the histidine residue from the linker, both resulted in improved membrane disruptive activity at the endosomal pH of 6.5. Meanwhile, the cellular uptake into HT29 cancer cells was improved, not only by increasing the amines of the head group, but also by increasing the degree of unsaturation in the lipid tails. Due to flexibility of the synthetic procedure, the delivery system could be modified further for different applications. The success of ECO and ECLn for in vitro siRNA delivery potentially makes them promising candidates for future in vivo studies.

Molecular MRI of atherosclerotic plaque progression in an ApoE(-/-) mouse model with a CLT1 peptide targeted macrocyclic Gd(III) chelate.

Molecular imaging of atherosclerotic biomarkers is critical for non-invasive detection and diagnosis of atherosclerotic plaques and therapeutic management. Fibrin and fibronectin accumulate at elevated levels in atherosclerotic plaques and are associated with atherogenesis and disease progression. Molecular imaging of these biomarkers has the potential to non-invasively characterize plaque burden. In this work, we investigated the effectiveness of a peptide-targeted macrocyclic Gd(III) chelate, CLT1-dL-(DOTA-Gd)4, specific to fibrin-fibronectin complexes for molecular MRI of atherosclerosis. Atherosclerotic plaques were induced in Apolipoprotein E-knockout (ApoE(-/-)) mice by feeding with high fat and cholesterol-enriched diet (HFD) for up to 30 weeks. MRI of the vessel wall in the arch aorta was performed at 10, 20 and 30 weeks after the onset of HFD. High spatial-resolution MRI was performed prior and up to 35 minutes after i.v. injection of CLT1-dL-(DOTA-Gd)4 or a nonspecific control agent at a dose of 0.1 mmol-Gd/kg. CLT1-dL-(DOTA-Gd)4 produced stronger enhancement in the atherosclerotic lesions of the aortic wall than the control at all time points in the mice. Cross sectional MR images of the aortic arch revealed progressive thickening of the atherosclerotic vessel wall in the mice on HFD for up to 30 weeks. This progression correlated well to histological staining, as well as fibrin and fibronectin immunochemical stained images. Molecular MRI with CLT1-dL-(DOTA-Gd)4 has a potential for detecting atherosclerosis and non-invasive monitoring of the progression of the plaques.

Albumin pre-coating enhances intracellular siRNA delivery of multifunctional amphiphile/siRNA nanoparticles.

Nonspecific association of serum molecules with short-interfering RNA (siRNA) nanoparticles can change their physiochemical characteristics, and results in reduced cellular uptake in the target tissue during the systemic siRNA delivery process. Serum albumin is the most abundant protein in the body and has been used to modify the surface of nanoparticles, to inhibit association of other serum molecules. Here, we hypothesized that surface modification of lipid-based nanoparticular siRNA delivery systems with albumin could prevent their interaction with serum proteins, and improve intracellular uptake. In this study, we investigated the influence of albumin on the stability and intracellular siRNA delivery of the targeted siRNA nanoparticles of a polymerizable and pH-sensitive multifunctional surfactant N-(1-aminoethyl) iminobis[N-(oleoylcysteinylhistinyl-1-aminoethyl)propionamide] (EHCO) in serum. Serum resulted in a significant increase in the size of targeted EHCO/siRNA nanoparticles and inhibited cellular uptake of the nanoparticles. Coating of targeted EHCO/siRNA nanoparticles with bovine serum albumin at 9.4 µM prior to cell transfection improved cellular uptake and gene silencing efficacy of EHCO/siRNA targeted nanoparticles in serum-containing media, as compared with the uncoated nanoparticles. At a proper concentration, albumin has the potential to minimize interactions of serum proteins with siRNA nanoparticles for effective systemic in vivo siRNA delivery.