Nitric oxide and other reactive nitrogen species target multiple sites in the mitochondria to impact cellular bioenergetics and survival. Kinetic imaging studies revealed that NO from either activated macrophages or donor compounds rapidly diffuses to the mitochondria, causing a dose dependent progressive increase in NO-dependent DAF fluorescence that corresponded to mitochondrial membrane potential loss, and initiated alterations in cellular bioenergetics that ultimately led to necrotic cell death. Cellular dysfunction is mediated by an elevated 3-nitrotyrosine signature of the mitochondrial complex I subunit NDUFB8, which is vital for normal mitochondrial function as evidenced by selective knockdown via siRNA. Overexpression of mitochondrial superoxide dismutase substantially decreased NDUFB8 nitration and restored mitochondrial homeostasis. Further, treatment of cells with either necrostatin-1 or siRNA knockdown of RIP1 and RIP3 prevented NO-mediated necrosis. This work demonstrates that the interaction between NO and mitochondrially-derived superoxide alters mitochondrial bioenergetics and cell function, thus providing a molecular mechanism for reactive oxygen and nitrogen species-mediated alterations in mitochondrial homeostasis.

This paper reviews the recent findings related to the physical properties of tetraether lipid membranes, with special attention to the effects of the number, position, and configuration of cyclopentane rings on membrane properties. We discuss the findings obtained from liposomes and monolayers, composed of naturally occurring archaeal tetraether lipids and synthetic tetraethers as well as the results from computer simulations. It appears that the number, position, and stereochemistry of cyclopentane rings in the dibiphytanyl chains of tetraether lipids have significant influence on packing tightness, lipid conformation, membrane thickness and organization, and headgroup hydration/orientation.

Combretastatin A4 disodium phosphate (CA4P) is a fluorescent, water-soluble prodrug able to induce vascular shutdown within tumors at doses less than one-tenth of the maximum tolerated dose. As a continued effort to develop efficient liposomal CA4P to treat solid tumor, we herein investigate the physical and spectroscopic properties of CA4P in aqueous solution and the mechanism of CA4P release from archaeal tetraether liposomes (archaeosomes). We found that cis-CA4P can be photoisomerized to trans-CA4P. This photoisomerization results in an increase in fluorescence intensity. Both cis- and trans-CA4P undergo fluorescence intensity self-quenching after they reach a critical concentration Cq (∼0.15-0.25 mM). Moreover, both cis- and trans-CA4P in buffer exhibit a red shift in their excitation spectrum and an increase in excitation spectrum band sharpness with increasing concentration, which can be attributed to the formation of J-aggregates. The onset of the dramatic change in excitation maximum occurs at concentrations close to Cq, suggesting that the self-quenching arises from extensive J-aggregate formation and that, when CA4P concentration exceeds Cq, J-aggregate formation begins to increase sharply. Our data also suggest that the extent of J-aggregate formation plays a critical role in CA4P release from tetraether archaeosomes and in the subsequent cytotoxicity on cultured human breast cancer MCF-7 cells. The drug leakage and cytotoxicity rate constants vary with the initial CA4P concentration entrapped inside archaeosomes in a biphasic manner, reaching a local maximum at 0.25-0.50 mM. A mechanism based on the concept of J-aggregate formation has been proposed to explain the biphasic changes in drug release and cytotoxicity with increasing drug concentration. Tetraether archaeosomes are extraordinarily stable and relatively nontoxic to animals; thus, they are promising nano drug carriers. The results obtained from this study pave the way for future development of archaeosomal CA4P to treat solid tumors.

Liposomes have many advantages as therapeutic capsules over free drugs such as small molecule drugs and nucleic acids. Cholesterol is commonly used as a membrane stabilizing agent in liposomal drugs (e.g., mRNA-lipid nanoparticle COVID-19 vaccines). However, due to the vulnerability of cholesterol to oxidation and the etiological role of cholesterol in many disorders, it is desirable to find an alternative means to stabilize liposomal membranes for drug delivery. In this study, we demonstrated that the polar lipid fraction E (PLFE), which contains exclusively bipolar tetraether macrocyclic lipids, isolated from the thermoacidophilic archaeon S. acidocaldarius can greatly stabilize the liposomal formulation of the anti-vascular drug, combretastatin A4 disodium phosphate (CA4P). Stability was assessed by determining the leakage rate constant k of entrapped CA4P fluorometrically. We found that, at 37 °C, PLFE decreases the k value monotonically from 1.54 × 10−2 s−1 for 100% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) liposomes to 3.4 × 10−5 s−1 for 100% PLFE archaeosomes, a change of k by two orders of magnitude. The changes in k of CA4P leakage are correlated well with the changes in liposomal CA4P’s cytotoxicity against MCF-7 breast cancer cells. We further showed that the reduction in spontaneous leakage of entrapped CA4P by PLFE can be attributed to the increased membrane surface charge and the increased membrane order and packing tightness in liposomes, as reflected by the zeta potential (−6.83 to −41.1 mV from 0 to 100 mol% PLFE) and diphenylhexatriene (DPH) fluorescence polarization (0.13 to 0.4 from 0 to 100 mol% PLFE) measurements. Moreover, we showed that PLFE slows down CA4P leakage more than cholesterol in POPC liposomes. These results together suggest that PLFE lipids can serve as an effective stabilizing agent for liposomal drugs and could potentially be useful for the optimization of liposomal CA4P for cancer treatment.

entrapment efficiency and spontaneous release of anti-cancer drug doxorubicin is studied by using absorption spectroscopy and fluorescence de-quenching method. These physical measurements are performed on liposomes with varying PLFE molar ratios at different temperatures. The obtained results may help to optimize and design liposomal drugs with greater stability and higher therapeutic efficacy. (supported by NSF DMR1105277)