Efficient electroporation of liposomes doped with pore stabilizing nisin.

CONTEXT: Liposomes have a long history as passive and active drug carriers. Recently, a few methods have been realized to control the release from liposomes, including heating, ultrasound and laser. OBJECTIVE: We report on a new approach to drive release from liposomes using electric fields. MATERIALS AND METHODS: Liposomes were manufactured containing a high concentration of (quenched) 5-6 carboxyfluorescein dye. Nisin, a well-known amphiphilic peptide lantibiotic that works by stabilizing pores formed in cell membranes, was mixed in solution inside or outside the liposomes. The liposomes were then electroporated using a range of voltages, and assayed for increases in fluorescence due to release of dye. Release was measured against positive and negative controls, with positive control release driven by a strong detergent. RESULTS: Our results demonstrate that the addition of nisin significantly reduces the electric field required to release the contents of liposomes, from 2000 V/m to approximately 200 V/m. This result proves that, in principle, electroporation (EP) of liposomes doped with small amounts of amphiphilic pore stabilizing peptides may be a practical means to drive release of liposomal contents in vivo. CONCLUSION: Drug delivery from liposomes doped with amphiphilic peptides using EP is feasible. This technique could be developed into a potent adjuvant to tumor ablation using irreversible EP.

Low-conductance HCN1 ion channels augment the frequency response of rod and cone photoreceptors.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are expressed in several tissues throughout the body, including the heart, the CNS, and the retina. HCN channels are found in many neurons in the retina, but their most established role is in generating the hyperpolarization-activated current, I(h), in photoreceptors. This current makes the light response of rod and cone photoreceptors more transient, an effect similar to that of a high-pass filter. A unique property of HCN channels is their small single-channel current, which is below the thermal noise threshold of measuring electronics. We use nonstationary fluctuation analysis (NSFA) in the intact retina to estimate the conductance of single HCN channels, revealing a conductance of approximately 650 fS in both rod and cone photoreceptors. We also analyze the properties of HCN channels in salamander rods and cones, from the biophysical to the functional level, showing that HCN1 is the predominant isoform in both cells, and demonstrate how HCN1 channels speed up the light response of both rods and cones under distinct adaptational conditions. We show that in rods and cones, HCN channels increase the natural frequency response of single cells by modifying the photocurrent input, which is limited in its frequency response by the speed of a molecular signaling cascade. In doing so, HCN channels form the first of several systems in the retina that augment the speed of the visual response, allowing an animal to perceive visual stimuli that change more quickly than the underlying photocurrent.

Complementary conductance changes by IKx and Ih contribute to membrane impedance stability during the rod light response.

In addition to the HCN1 channels that mediate the h current, the Kx current also performs signal filtering in rod photoreceptors. This current is known to be mediated by potassium channels and has similarities to the neuronal M current and EAG potassium channels. Although it is known that in filtering the light response of rods, I(h) and I(Kx) undergo complementary conductance changes, the qualities and significance of these changes are not clear. Here we present an analysis demonstrating the filtering effect of HCN1 channels in salamander rods when I(Kx) is blocked, and a simulation of the rod light response showing the magnitude and time course of the conductance changes by both currents. From this analysis, we propose that the purpose of opposing conductance changes by I(h) and I(Kx) may be to optimize the lateral propagation of signals through gap junctions in the rod network.

Genetic dissection of rod and cone pathways in the dark-adapted mouse retina.

A monumental task of the mammalian retina is to encode an enormous range (>10(9)-fold) of light intensities experienced by the animal in natural environments. Retinal neurons carry out this task by dividing labor into many parallel rod and cone synaptic pathways. Here we study the operational plan of various rod- and cone-mediated pathways by analyzing electroretinograms (ERGs), primarily b-wave responses, in dark-adapted wildtype, connexin36 knockout, depolarizing rod-bipolar cell (DBCR) knockout, and rod transducin alpha-subunit knockout mice [WT, Cx36(-/-), Bhlhb4(-/-), and Tralpha(-/-)]. To provide additional insight into the cellular origins of various components of the ERG, we compared dark-adapted ERG responses with response dynamic ranges of individual retinal cells recorded with patch electrodes from dark-adapted mouse retinas published from other studies. Our results suggest that the connexin36-mediated rod-cone coupling is weak when light stimulation is weak and becomes stronger as light stimulation increases in strength and that rod signals may be transmitted to some DBCCs via direct chemical synapses. Moreover, our analysis indicates that DBCR responses contribute about 80% of the overall DBC response to scotopic light and that rod and cone signals contribute almost equally to the overall DBC responses when stimuli are strong enough to saturate the rod bipolar cell response. Furthermore, our study demonstrates that analysis of ERG b-wave of dark-adapted, pathway-specific mutants can be used as an in vivo tool for dissecting rod and cone synaptic pathways and for studying the functions of pathway-specific gene products in the retina.

Impedance measurements for cervical cancer diagnosis.

This article discusses using impedance measurements of body tissue in a diagnostic device. It then reviews the theory behind using these measurements to separate normal from diseased tissue. A small amount of time is devoted to discussing the meaning of sensitivity, specificity, and the receiver operating characteristic (ROC) curve, and their meanings. It also discusses the prospects of some new clinical devices using impedance measurements. One of the devices it focuses on is the TruScreen probe made by the Australian firm, Polartechnics.