Stephen W. Jones

Using mechanobiological mimicry of red blood cells to extend circulation times of hydrogel microparticles

It has long been hypothesized that elastic modulus governs the biodistribution and circulation times of particles and cells in blood; however, this notion has never been rigorously tested. We synthesized hydrogel microparticles with tunable elasticity in the physiological range, which resemble red blood cells in size and shape, and tested their behavior in vivo. Decreasing the modulus of these particles altered their biodistribution properties, allowing them to bypass several organs, such as the lung, that entrapped their more rigid counterparts, resulting in increasingly longer circulation times well past those of conventional microparticles. An 8-fold decrease in hydrogel modulus correlated to a greater than 30-fold increase in the elimination phase half-life for these particles. These results demonstrate a critical design parameter for hydrogel microparticles.

PEGylated PRINT nanoparticles: The impact of PEG density on protein binding, macrophage association, biodistribution, and pharmacokinetics

In this account, we varied PEGylation density on the surface of hydrogel PRINT nanoparticles and systematically observed the effects on protein adsorption, macrophage uptake, and circulation time. Interestingly, the density of PEGylation necessary to promote a long-circulating particle was dramatically less than what has been previously reported. Overall, our methodology provides a rapid screening technique to predict particle behavior in vivo and our results deliver further insight to what PEG density is necessary to facilitate long-circulation.

Overview of Voltage-Dependent Calcium Channels

Voltage-dependent calcium channels couple electrical signals to cellular responses in excitable cells. Calcium channels are crucial for excitation–secretion coupling in neurons and endocrine cells, and excitation–contraction coupling in muscle. Several pharmacologically and kinetically distinct calcium channel types have been identified at the electrophysiological and molecular levels. This review summarizes the basic properties of voltage-dependent calcium channels, including mechanisms of ion permeation and block, gating kinetics, and modulation by G proteins and second messengers.

Calcium currents in the A7r5 smooth muscle-derived cell line

We have studied voltage-dependent calcium channels in the A7r5 smooth muscle cell line by measuring the high-affinity binding of radiolabelled dihydropyridines (DHPs), whole-cell and single-channel currents in patchclamped cells, as well as cytosolic calcium ([Ca2+]i) in fura-2-loaded cell suspensions and monolayers. Intact A7r5 cells express saturable, high-affinity, voltage-sensitive DHP binding sites with pharmacological properties characteristic of L-type calcium channels. When cells were voltage clamped in the whole-cell configuration with near normal intra- and extracellular solutions, a DHP-sensitive inward current resembling the L-type calcium current was dominant. With barium (10 mM) as the charge carrier, peak inward currents were typically recorded at test potentials between 0 and +20 mV. Currents were blocked by extracellular cadmium with a half-maximal inhibitory concentration of ∼ 1 μM. Isoproterenol (1 μM.) or forskolin (10 μM) increased currents in approximately half of the cells tested. Forskolin (10 μM) increased single-channel activity in five of eight cell-attached patches. After cells had been quiescent for several weeks, cell suspensions showed changes in resting [Ca2+]i in response to DHPs and increased potassium. Most confluent monolayers of cells showed spontaneous transient elevations in [Ca2+]i. Bath application of Bay K 8644 increased the frequency and magnitude of these [Ca2+]i transients, whereas nifedipine abolished the transients. These data suggest that the [Ca2+]i transients were due to synchronous action potentials in electrically coupled cell monolayers.

Dihydropyridine actions on calcium currents of frog sympathetic neurons

Dihydropyridines (DHPs) generally have little effect on whole-cell calcium currents of neurons, even at concentrations far higher than those effective on muscle. Either neuronal calcium currents are much less sensitive to DHPs, or only a small proportion of the current is DHP-sensitive. We find that DHP agonists and antagonists act at low concentration on calcium currents in frog sympathetic neurons but that the effects are small even at optimal concentrations. The half-maximal dose (EC50) of the agonist Bay K 8644 is approximately 50 nM, and the effect of Bay K 8644 is blocked by 50% at approximately 300 nM nifedipine, from a holding potential of -80 mV. Nifedipine is more effective from a holding potential of -50 mV. These results suggest the presence of an L-type calcium current, with DHP sensitivity similar to L-currents in cardiac muscle. The predominant (greater than 90%) calcium current in frog sympathetic neurons is a DHP-resistant N-type current. However, high concentrations of DHPs (10 microM) partially block N-type calcium current, as well as voltage-dependent sodium and potassium currents.

Muscarinic and peptidergic excitation of bull‐frog sympathetic neurones.

The large B cells of bull‐frog sympathetic ganglia are well known to be depolarized by slow synaptic transmission, muscarinic agonists, analogues of luteinizing hormone‐releasing hormone (LHRH), and substance P. Voltage‐clamp analysis shows that these actions result from two underlying mechanisms: inhibition of the M‐current, a voltage‐dependent potassium current; and in some cells, an inward current associated with an increase in conductance. The additional inward current appears as a voltage‐insensitive change in the instantaneous conductance (i.e. apparent leak conductance). The additional inward current is typically slower in onset and offset than is M‐current inhibition. It is typically seen for higher concentrations and longer durations of agonist application. In many cells, only a decrease in M‐current can be demonstrated. Muscarine inhibits the M‐current with 50% inhibition (I50) at 0.7 microM. At least 86% of the M‐current is muscarine sensitive. At comparable concentrations, oxotremorine produces less M‐current inhibition than does muscarine. Some analogues of teleost LHRH (T‐LHRH) are more potent as M‐current inhibitors than T‐LHRH itself. Those peptides tend to act more slowly than T‐LHRH. Substance P shows variable potency for M‐current inhibition, with I50 s ranging from 2 nM to greater than 2 microM on different cells. The response to long applications of substance P desensitizes in some cells, which has not been observed for comparable applications of muscarinic or LHRH agonists. Other tachykinins (including substance K) inhibit the M‐current. C‐terminal fragments of substance P are ineffective, and M‐current inhibition by substance P is not blocked by [D‐Pro2,D‐Trp7,9]‐ or [D‐Arg1,D‐Pro2, D‐Trp7,9,Leu11] substance P. The slow muscarinic excitatory post‐synaptic potential (e.p.s.p.) produces a graded inhibition of up to 90% of the M‐current. Occasional cells show an additional inward current with an associated increase in conductance during the slow e.p.s.p. This effect is less marked than with exogenous muscarinic agonists. The late, slow e.p.s.p., which is produced by stimulation of high threshold C‐fibre inputs and is resistant to cholinergic antagonists, also involves M‐current inhibition. An additional inward current can be observed in some cells. M‐current inhibition (by agonists or slow synaptic potentials) increases the number of spikes produced by a given depolarizing current, often allowing maintained firing. This action is not mimicked by equivalent depolarization, and is still seen when the cell is manually clamped to the original resting potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Nanoparticle clearance is governed by Th1/Th2 immunity and strain background

Extended circulation of nanoparticles in blood is essential for most clinical applications. Nanoparticles are rapidly cleared by cells of the mononuclear phagocyte system (MPS). Approaches such as grafting polyethylene glycol onto particles (PEGylation) extend circulation times; however, these particles are still cleared, and the processes involved in this clearance remain poorly understood. Here, we present an intravital microscopy-based assay for the quantification of nanoparticle clearance, allowing us to determine the effect of mouse strain and immune system function on particle clearance. We demonstrate that mouse strains that are prone to Th1 immune responses clear nanoparticles at a slower rate than Th2-prone mice. Using depletion strategies, we show that both granulocytes and macrophages participate in the enhanced clearance observed in Th2-prone mice. Macrophages isolated from Th1 strains took up fewer particles in vitro than macrophages from Th2 strains. Treating macrophages from Th1 strains with cytokines to differentiate them into M2 macrophages increased the amount of particle uptake. Conversely, treating macrophages from Th2 strains with cytokines to differentiate them into M1 macrophages decreased their particle uptake. Moreover, these results were confirmed in human monocyte-derived macrophages, suggesting that global immune regulation has a significant impact on nanoparticle clearance in humans.

Substance P inhibits the M‐current in bullfrog sympathetic neurones

Substance P (SP, 2.5–10 μm) was applied by rapid bath perfusion to bullfrog lumbar sympathetic neurones in vitro, voltage‐clamped through a single micro‐electrode. In unclamped cells, SP produced a depolarization accompanied by an increase in apparent input resistance. Under voltage‐clamp a voltage‐dependent inward current was induced by SP, during which the time‐dependent relaxations induced by square voltage commands were inhibited. It is concluded that SP inhibits the M‐current (IM), a species of voltage‐dependent K+‐current, and that IM‐inhibition was the primary cause of the inward current and membrane depolarization in the cells tested.

Human TRPC6 expressed in HEK 293 cells forms non-selective cation channels with limited Ca2+ permeability

TRPC6 is thought to be a Ca2+‐permeable cation channel activated following stimulation of G‐protein‐coupled membrane receptors linked to phospholipase C (PLC). TRPC6 current is also activated by exogenous application of 1‐oleoyl‐acetyl‐sn‐glycerol (OAG) or by inhibiting 1,2‐diacylglycerol (DAG) lipase activity using RHC80267. In the present study, both OAG and RHC80267 increased whole‐cell TRPC6 current in cells from a human embryonic kidney cell line (HEK 293) stably expressing TRPC6, but neither compound increased cytosolic free Ca2+ concentration ([Ca2+]i) when the cells were bathed in high‐K+ buffer to hold the membrane potential near 0 mV. These results suggested that TRPC6 channels have limited Ca2+ permeability relative to monovalent cation permeability and/or that Ca2+ influx via TRPC6 is greatly attenuated by depolarization. To evaluate Ca2+ permeability, TRPC6 currents were examined in extracellular buffer in which Ca2+ was varied from 0.02 to 20 mm. The results were consistent with a pore‐permeation model in which Ca2+ acts primarily as a blocking ion and contributes only a small percentage (∼4%) to whole‐cell currents in the presence of extracellular Na+. Measurement of single‐cell fura‐2 fluorescence during perforated‐patch recording of TRPC6 currents showed that OAG increased [Ca2+]i 50–100 nm when the membrane potential was clamped at between −50 and −80 mV, but had little or no effect if the membrane potential was left uncontrolled. These results suggest that in cells exhibiting a high input resistance, the primary effect of activating TRPC6 will be membrane depolarization. However, in cells able to maintain a hyperpolarized potential (e.g. cells with a large inwardly rectifying or Ca2+‐activated K+ current), activation of TRPC6 will lead to a sustained increase in [Ca2+]i. Thus, the contribution of TRPC6 current to both the kinetics and magnitude of the Ca2+ response will be cell specific and dependent upon the complement of other channel types.

Sodium currents in dissociated bull‐frog sympathetic neurones.

1. Sodium currents were recorded from the cell bodies of single dissociated sympathetic neurones of bull‐frogs, using patch electrodes in the whole‐cell configuration, in Cs+‐loaded cells, using external Mn2+ to block calcium currents. 2. A discontinuous single‐electrode voltage‐clamp method was used. Switching frequencies of 40‐50 kHz were possible with 0.5‐2 M omega electrodes, giving clamp settling times of approximately 0.2 ms and adequate clamp of currents up to 30 nA. 3. The sodium currents required unusually positive voltages for both activation and inactivation, with half of the maximal observed conductance activating at +2 mV, and half‐maximal steady‐state inactivation at ‐35 mV. Both fast (in the order of milliseconds) and slow (in the order of seconds) inactivation processes occurred. 4. Two pharmacologically and kinetically distinct sodium currents were observed. The larger current was blocked by tetrodotoxin (TTX) and saxitoxin (STX) with I50 values (i.e. the concentration which results in 50% inhibition) of 10 nM or lower, and activated and inactivated relatively rapidly. 5. A smaller current (approximately 25% of peak current) was blocked by 0.1‐1 microM‐STX but not by 1‐10 microM‐TTX. It also activated rapidly, but inactivated approximately 3‐fold more slowly than the larger current. The slower current was blocked 75‐90% by Cd2+ (50‐200 microM).