Transdermal delivery of timolol by electroporation through human skin.

The purpose was to achieve therapeutic fluxes of timolol by transdermal delivery using skin electroporation. The transdermal transport of timolol through human stratum corneum was studied in three compartment diffusion cells. The electrodes, buffer composition and pulse conditions were optimized. Timolol maleate concentration in the donor compartment was 40 mg/ml. Square wave pulses were applied. Electroporation enhanced the transdermal transport of timolol by 1-2 orders of magnitude as compared to passive diffusion. Even though the current application lasted for only 10 s, the transdermal transport remained high after pulsing for at least 6 h. Higher fluxes were obtained with Pt electrodes close to the skin and a phosphate buffer. 10 pulses of 400 V-10 ms were more efficient than 10 low voltage-long duration pulses. Therapeutic fluxes of timolol (>50 microg/cm(2) per h) through human stratum corneum were achieved by electroporation.     

DNA electrotransfer into the skin using a combination of one high- and one low-voltage pulse.

Electroporation is an effective alternative to viral methods to significantly improve DNA transfection after intradermal and topical delivery. The aim of the study was to check whether a combination of a short high-voltage pulse (HV) to permeabilize the skin cells and a long low-voltage pulse (LV) to transfer DNA by electrophoresis was more efficient to enhance DNA expression than conventional repeated HV or LV pulses alone after intradermal injection of DNA plasmid. GFP and luciferase expressions in the skin were enhanced by HV+LV protocol as compared to HV or LV pulses alone. The expression lasted for up to 10 days. Consistently, HV+LV protocol induced a higher Th2 immune response against ovalbumin than HV or LV pulses. Standard methods were used to assess the effect of electric pulses on skin: the application of a combination of HV and LV pulses on rat skin fold delivered by plate electrodes was well tolerated. These data demonstrate that a combination of one HV (700 to 1000 V/cm; 100 micros) followed by one LV (140 to 200 V/cm; 400 ms) is an efficient electroporation protocol to enhance DNA expression in the skin.     

Transdermal delivery of fentanyl: rapid onset of analgesia using skin electroporation

Skin electroporation has recently been shown to increase transdermal transport of small-size drugs as well as considerably larger molecules by up to 4 orders of magnitude in vitro. Nevertheless, no in vivo studies have proven that high-voltage pulses can induce therapeutic plasma levels of drug. The aim of the present report was precisely to study the potential of skin electroporation in transdermal delivery of fentanyl in vivo. Fentanyl was transdermally delivered to hairless rats using high-voltage pulsing. Following the administration, the pharmacokinetics and pharmacodynamics were assessed. Significant fentanyl plasma concentrations were rapidly achieved using skin electroporation. Immediately after the 5 min pulsing, fentanyl plasma levels reached one third of the maximal plasma concentration of 30 ng/ml, the peak occurring 30 min after the electroporation. Deep analgesia and supraspinal effects were achieved, antinociception lasting for an hour. The magnitude of the effects was, however, dependent on the electrical parameters of the pulses.     

In vivo assessment of skin electroporation using square wave pulses.

The application of short-duration high-voltage pulses to the skin has been shown to enhance transdermal drug delivery by several orders of magnitude and to transiently permeabilize cells in tissue. Both exponentially decaying (ED) pulses and square wave (SW) pulses have been applied. The latter have also been used for electrochemotherapy. To date, their effect on skin integrity has not been analyzed. The scope of this work was (i) to investigate the effect induced by SW pulses on the stratum corneum and the skin, (ii) to evaluate the safety issue associated with electroporation, (iii) to contribute to the understanding of drug transport. Biophysical techniques (transepidermal water loss, chromametry, impedance and laser Doppler velocimetry or imaging measurement) and histological methods were combined to provide a global picture of the effects. Ten SW pulses applied to the skin induced a mild impairment of the skin barrier function and a dramatic decrease in skin resistance. These changes were reversible. A transient decrease (<5 min) in blood flow was observed. Neither inflammation, nor necroses were observed. These studies confirm the tolerance of the skin to square wave pulses in vivo.     

Electrophoretic component of electric pulses determines the efficacy of in vivo DNA electrotransfer

Efficient DNA electrotransfer can be achieved with combinations of short high-voltage (HV) and long low voltage (LV) pulses that cover two effects of the pulses, namely, target cell electropermeabilization and DNA electrophoresis within the tissue. Because HV and LV can be delivered with a lag up to 3000 sec between them, we considered that it was possible to analyze separately the respective importance of the two types of effects of the electric fields on DNA electrotransfer efficiency. The tibialis cranialis muscles of C57BL/6 mice were injected with plasmid DNA encoding luciferase or green fluorescent protein and then exposed to various combinations of HV and LV pulses. DNA electrotransfer efficacy was determined by measuring luciferase activity in the treated muscles. We found that for effective DNA electrotransfer into skeletal muscles the HV pulse is prerequisite; however, its number and duration do not significantly affect electrotransfer efficacy. DNA electrotransfer efficacy is dependent mainly on the parameters of the LV pulse(s). We report that different LV number, LV individual duration, and LV strength can be used, provided the total duration and field strength result in convenient electrophoretic transport of DNA toward and/or across a permeabilized membrane.     

Transdermal delivery of cyclosporin-A using electroporation

Topical delivery of cyclosporin A (CSA) is desirable for treating psoriasis, but it is hindered by the barrier property of stratum corneum, and the physicochemical properties of CSA. Attempts to deliver CSA from a solution prepared in 40% ethanol (EtOH) in phosphate buffered saline (PBS) using iontophoresis did not result in any significant increase in drug delivery, compared to passive. However, the use of electroporation pulses as a physical penetration enhancer enabled delivery of a significant amount of CSA. Single pulse electroporation study indicated that the amount of EtOH delivered across the skin increased as the applied electrode voltage (U_electrode) was increased. However, it did not translate into a proportional increase in the delivery of CSA and only a three to four times increase, compared to passive delivery, was seen with the single pulse electroporation. The drug contact duration had a varying effect in the efficiency of transdermal delivery of CSA. Four hour contact duration was chosen for the multiple pulse study. Use of multiple pulses (25 pulses, 10 ms each) at U_electrode 200 V resulted in a sixty-fold increase, compared to passive, in the delivery of CSA to the skin. Transdermally delivered CSA was mostly bound to the skin and only a small amount was seen to cross the full skin into the receiver compartment. In a study of solvent transport, the flux of water was up to three times larger than that of EtOH after electroporation.     

Spatially constrained localized transport regions due to skin electroporation.

Rapid, controlled molecular transport across human skin is of great interest for transdermal drug delivery and minimally invasive chemical sensing. Short, high-voltage pulses have been shown previously to create localized transport regions in the skin. Here, we show that these regions can be constrained to occur at specific sites using electrically insulating masks that restrict the field lines. The increase in total ionic and molecular transport per area was comparable to the levels observed in unconstrained electroporation of human skin. Constraining the area of intervention to encompass small areas of interest, a primary feature in the design of microdevices for transdermal drug delivery, can provide the same levels of flux as the unconstrained case.     

Do high-voltage pulses cause changes in skin structure?

Dramatic changes in skin properties are caused by high-voltage pulses (e.g., 100 V) of millisecond duration. The exact mechanism by which these changes occur remains unresolved, but may result from alterations of skin structure, possibly involving electroporation of the stratum corneum's intracellular lipid bilayers. The evidence supporting this hypothesis is presented from a range of studies which address molecular transport, electrical impedance, microscopic imaging and theoretical analysis.     

The pulse length-dependence of inertial cavitation dose and hemolysis

Gas-based ultrasound (US) contrast agents increase erythrocyte sonolysis, presumably via enhancing inertial cavitation (IC) activity. The amount of IC activity (IC "dose") and hemolysis generated by exposure to 1.15 MHz US were examined with different US pulse lengths, but with the same delivered acoustic energy, for Optison and Albunex. The hypotheses were that 1. at longer pulse lengths, IC would generate more bubbles that could nucleate additional IC activity; 2. if the interval between pulse pairs were short enough for the next pulse to hit derivative bubbles before their dissolution, more IC could be induced; and 3. hemolysis would be proportional to IC activity. Two types of studies were performed. In the first, bubble generation after each burst of IC activity was quantified using an active cavitation detector (ACD), for different pulse lengths (5, 10, 20, 30, 50, 100 or 200 cycles), but the same pressure level (3 MPa) and total "on" time (173.16 ms). Low concentrations of either Optison or Albunex were added into the tank with high-intensity and interrogating transducers orthogonal to each other. For pulse lengths > 100 cycles, and pulse repetition intervals < 5 ms, a "cascade" effect (explosive bubble generation) was observed. In the second, IC was measured by passive detection methods. IC dose and hemolysis were determined in whole blood samples at a pressure level (3 MPa) and interpulse interval (5 ms) that induced the "cascade" effect. Each blood sample was mixed with the same number of contrast microbubbles (Optison approximately 0.3 v/v % and Albunex approximately 0.5 v/v %), but exposed to different pulse lengths (5, 10, 20, 30, 50, 100 or 200 cycles). With Optison, up to 60% hemolysis was produced with long pulses (100 and 200 cycles), compared with < 10% with short pulses (5 and 10 cycles). Albunex generated considerably less IC activity and hemolysis. The r(2) value was 0.99 for the correlation between hemolysis and IC dose. High pulse-repetition frequency (PRF) (500 Hz) generated more hemolysis than the low PRF (200 Hz) at 3 MPa. All experimental results could be explained by the dissolution times of IC-generated bubbles.     

Cutaneous pain and detection thresholds to short CO2 laser pulses in humans: evidence on afferent mechanisms and the influence of varying stimulus conditions

Pain and detection thresholds to short CO2 laser pulses were studied in healthy human subjects. Pain thresholds were significantly higher than detection thresholds in both hairy and glabrous skin; in the glabrous skin both thresholds were higher in the hairy skin. The range from detection threshold to pain threshold was larger in the glabrous skin. The minimal energy per surface area needed to produce any sensation (detection) or pain sensation decreased with increasing stimulus surface, and this spatial summation effect was to equal magnitude in the hairy and the glabrous skin. With decreasing stimulus pulse duration (from 45 to 15 msec) the detection and pain thresholds were elevated: this effect was stronger on pain thresholds. With increasing adapting skin temperature, less energy was needed to produce any sensation (detection) or pain sensation. The effect of adapting skin temperature was equal on pain and detection thresholds. The conduction velocity of fibers mediating laser evoked first sensations was in the thin fiber range (less than 10 msec), according to a reaction time study. The results suggest that short CO2 laser pulses produce both non-pain and pain sensations, but that both these sensations are based on the activation of the same primary afferent fiber population of slowly conducting nociceptive fibers. Central summation of primary afferent impulses is needed to elicit a liminal non-painful sensation, and an increased number of impulses in the same fibers produces pain.     

Electroporation-mediated delivery of 3'-protected phosphodiester oligodeoxynucleotides to the skin.

The feasibility of topical delivery in the skin of 3' end modified phosphodiester oligonucleotides using electroporation was investigated. Experiments were performed in vitro, using hairless rat skin. Five pulses of (200 V, 450 ms) were applied. The 3' end modifications of the 15 mer oligonucleotide were: (1) 3'-aminohexyl, (2) biotin, with a triethyleneglycol arm, (3) methylphosphonate links between nucleotides 13, 14 and 15, and (4) 2-O-methyl nucleotides at 13, 14 and 15 positions. All the modifications were efficient to protect the oligonucleotides against degradation in the skin. Electroporation increased the topical delivery of the 3' end-modified phosphodiesters by two orders of magnitude compared to passive diffusion, without significant differences between the derivatives. Oligonucleotide concentrations in the range of 1 microm could be achieved in the viable skin. The delivery of a phosphorothioate congener was lower than phosphodiester delivery due to the interaction of phosphorothioate with the stratum corneum. Consequently, 3' end-protected phosphodiesters could be an interesting alternative to phosphorothioate oligonucleotides for topical treatment of cutaneous diseases.     

A Theoretical Study of Inertial Cavitation from Acoustic Radiation Force Impulse Imaging and Implications for the Mechanical Index

The mechanical index (MI) attempts to quantify the likelihood that exposure to diagnostic ultrasound will produce an adverse biological effect by a non-thermal mechanism. The current formulation of the MI implicitly assumes that the acoustic field is generated using the short pulse durations appropriate to B-mode imaging. However, acoustic radiation force impulse (ARFI) imaging employs high-intensity pulses up to several hundred acoustic periods long. The effect of increased pulse durations on the thresholds for inertial cavitation was studied computationally in water, urine, blood, cardiac and skeletal muscle, brain, kidney, liver and skin. The results indicate that, although the effect of pulse duration on cavitation thresholds in the three liquids can be considerable, reducing them by, for example, 6%–24% at 1 MHz, the effect on tissue is minor. More importantly, the frequency dependence of the MI appears to be unnecessarily conservative; that is, the magnitude of the exponent on frequency could be increased to 0.75. Comparison of these theoretical results with experimental measurements suggests that some tissues do not contain the pre-existing, optimally sized bubbles assumed for the MI. This means that in these tissues, the MI is not necessarily a strong predictor of the probability of an adverse biological effect.     

AOPT强脉冲光在轻度面部皮肤松弛中的应用

目的 观察第七代强脉冲光AOPT对面部轻度皮肤松弛的治疗效果.方法 治疗前清洁面治疗区域,给予强脉冲光治疗(科医人公司M22第七代AOPT).光斑大小3.5cm×1.5cm.640nm滤光片,脉宽15～18ms,三脉冲,时间延迟30～35ms,能量15～19J/cm2,Vascular滤光片,脉宽12～15ms,双脉冲...     

Effect of microbubble fragility on transit rate measurement by contrast echography

We sought to propose a simplified method to measure flow velocity based on ultrasonic microbubble destruction, and investigated the effect of microbubble shell fragility on such measurement. Acoustic density (AD) from the second harmonic short axis image of flow was obtained at variable velocities (2 to 73 mm/s) in an in vitro model during long (1000 ms) and short (33 ms) interval ultrasound (US) pulsing, allowing complete and partial microbubble replenishment between pulses, respectively. Microbubbles with shell elastic modulus of 0.4 MPa and 16 MPa were tested. By shortening pulsing interval, AD diminished gradually, rather than abruptly, to a plateau level for both microbubbles. The extent of AD decay was greater for the fragile than the strong microbubbles. A linear relationship existed between the magnitude of AD decay and flow velocity only in the higher and lower velocity range for the fragile and the strong microbubbles, respectively. Thus, difference in contrast intensities during long and short pulsing intervals, respectively, allowing complete and partial replenishment may provide for velocity measurement, in which choice of optimal microbubble fragility for the range of velocity to measure may increase the accuracy.     

A physical mechanism to explain the delivery of chemical penetration enhancers into skin during transdermal sonophoresis - Insight into the observed synergism

The synergism between low-frequency sonophoresis (LFS) and chemical penetration enhancers (CPEs), especially surfactants, in transdermal enhancement has been investigated extensively since this phenomenon was first observed over a decade ago. In spite of the identifying that the origin of this synergism is the increased penetration and subsequent dispersion of CPEs in the skin in response to LFS treatment, to date, no mechanism has been directly proposed to explain how LFS induces the observed increased transport of CPEs. In this study, we propose a plausible physical mechanism by which the transport of all CPEs is expected to have significantly increased flux into the localized-transport regions (LTRs) of LFS-treated skin. Specifically, the collapse of acoustic cavitation microjets within LTRs induces a convective flux. In addition, because amphiphilic molecules preferentially adsorb onto the gas/water interface of cavitation bubbles, amphiphiles have an additional adsorptive flux. In this sense, the cavitation bubbles effectively act as carriers for amphiphilic molecules, delivering surfactants directly into the skin when they collapse at the skin surface as cavitation microjets. The flux equations derived for CPE delivery into the LTRs and non-LTRs during LFS treatment, compared to that for untreated skin, explain why the transport of all CPEs, and to an even greater extent amphiphilic CPEs, is increased during LFS treatment. The flux model is tested with a non-amphiphilic CPE (propylene glycol) and both nonionic and ionic amphiphilic CPEs (octyl glucoside and sodium lauryl sulfate, respectively), by measuring the flux of each CPE into untreated skin and the LTRs and non-LTRs of LFS-treated skin. The resulting data shows very good agreement with the proposed flux model.     

Electroporation-enhanced gene delivery in mammary tumors

Electroporation was applied to enhance gene transfer into subcutaneous MC2 murine breast tumors. Cultured MC2 cells were also transfected by electroporation or by cationic liposomes in the presence of serum using pSV-luc plasmids. Electroporation parameters and liposome formulation were optimized to achieve the highest relative levels of transfection. An electric field threshold for successful electrotransfection in cultured cells appeared around 800-900 V/cm. The liposomes used contained the cationic lipid dioleoyl-3-trimethylammonium propane (DOTAP). Multilamellar vesicles (MLV) had a 10-fold advantage over small unilamellar vesicles (SUV) in cell culture transfection. For in vivo gene delivery, the plasmids were injected either alone, or in complex with MLV or SUV DOTAP liposomes. A series of six electric pulses 1 ms long were applied across tumors, using caliper electrodes on the skin surface. Electric field strengths ranged from 400-2300 V/cm. Luciferase expression was approximately two orders of magnitude higher than controls in tumors treated with pulses > or =800 V/cm. Differences between enhanced relative levels of transfection using uncomplexed plasmid and lipoplexes were not statistically significant. Distribution of DNA into tumor tissues was monitored by fluorescence in situ PCR. The highest numbers of fluorescent cells were found in tumors electroporated following the injection of plasmid. The significant transfection improvement shows that in vivo electroporation is a powerful tool for local gene delivery to tumors.     

Internal Ventricular Defibrillation with Sequential Pulse Countershock in Pigs: Comparison with Single Pulses and Effects of Pulse Separation

We compared single to sequential pulse shocks with different pulse separations on internal cardiac defibrillation by using a catheter and plaque electrodes in open-chest halothane-anesthetized pigs. Ten seconds after fibrillation onset, defibrillation was attempted using trapezoidal pulses of 65% tilt, approximately 5 ms duration and fixed outputs from 1.0 to 50 joules (J). With single pulses, minimum defibrillation energy for the catheter alone was 2.4 ± 0.3 J/kg (mean ± standard error) and 2.1 ± 0.2 J/kg for the catheter tip to plaque configuration. With sequential pulse shocks, the first pulse delivered via the catheter and the second pulse from the catheter tip to the plaque electrode, the energy necessary for defibrillation was dependent on the separation time between the two pulses (2.0 ± 0.2, 1.5 ± 0.2, 0.9 ± 0.1, 1.3 ± 0.3, 0.6 ± 0.2, and 1.2 ± 0.2 J/kg at 100, 10, 1, 0.5, 0.2, and 0.1 ms, respectively). Further, at the 0.2 ms separation, 100% of the animals could be defibrillated with less than 2.0 J/kg (35 J total). We conclude that sequential pulse defibrillation provides a significant improvement over single pulse defibrillation. The optimum separation between the sequential pulses in this study was 0.2 ms.     

The effect of multiple stimuli on the modulation of the 'nociceptive' blink reflex

The 'nociceptive' blink reflex is a method of examining human trigeminal pain pathways. We explored temporal summation of this reflex by using a train of pulses, rather than a single pulse, and remote activation of diffuse noxious inhibitory control (DNIC), to improve reliability, flexibility and nociceptive specificity of this technique. The R2 component of the nociceptive blink reflex response (nR2) was assessed in 28 healthy volunteers using between 1 and 7 pulses per stimulus train (inter-pulse interval 5 ms). The effect of DNIC on single-, double-, and triple-pulse nR2 was investigated. Compared to single pulses, double and triple pulses increased the sensation of pain, reduced the tactile and pain thresholds, and facilitated the blink reflex responses (reduced onset latency, increased magnitude and persistence of nR2). The maximal reflex facilitation was achieved using a triple pulse. Higher pulse numbers had no additional facilitatory effect. Activation of the DNIC system using heterotopic pain suppressed the nR2 evoked by double and triple stimulation by 16 and 42%, respectively, but not the nR2 from a single pulse. Stimulation with double and triple pulses may be more suitable to study influences on nociceptive pathways than single pulses and may widen the methodological flexibility of the nociceptive blink reflex technique. This technique may be useful in studying the trigeminal nociceptive system with particular reference to primary headache disorders and their neuropharmacology.     

High intensity focused ultrasound ablation of kidney guided by MRI

The effectiveness of magnetic resonance imaging (MRI) to monitor therapeutic protocols of high-intensity focused ultrasound (HIFU), in freshly excised pig kidney cortex is investigated. For high quality imaging, the pulse sequence fast spin echo (FSE) T1- and T2-weighted, and proton density were evaluated. For fast imaging, the pulse sequence T1-weighted fast spoiled gradient (FSPGR) was used. The main goal was to evaluate the MRI detection of large lesions (bigger than 1 cm x 1 cm x 1 cm) that is achieved by moving the transducer in a predetermined pattern. The contrast between lesion and kidney tissue is excellent with either T1-weighted or T2-weighted FSE. With T1-weighted FSE, the best contrast is observed for recovery time (TR) between 200 ms and 400 ms. With T2-weighted FSE best contrast can be achieved for echo time (TE) between 16 and 32 ms. T2-weighted FSE was proven as the best pulse sequence to detect cavitational activity. This advantage is attributed to the significant difference in signal intensity between air spaces and necrotic tissue. Air spaces appear brighter than thermal lesions. Therefore, for therapeutic protocols created using cavitational mode, T2-weighted FSE may be the optimum pulse sequence to use. The proton density pulse sequence does not provide any advantage over the T1- and T2-weighted pulse sequences. Using T1-weighted FSPGR, acquisition time as low as 5 s could be achieved. Good contrast and signal-to-noise ratio (SNR) are achieved with TR = 100 ms and flip angle between 75 to 90 degrees. The above techniques were very successful in detecting large lesion volumes.     

The interaction of broadband intense pulsed light (IPL) and skin during a multi-pulse application

Intense pulsed light (IPL) systems are being presented as an alternative to conventional laser therapy for the treatment of vascular lesions. Broad spectral emission, radiant exposure, and pulse time are the key parameters for any IPL system used in clinical practice. Herein, we present the concept of using multi-pulse delivery to improve the vascular damage caused by IPL light.

The result of our animal study shows an increase in tissue temperature that depends on the number of applied IPL pulses. However, this temperature increase is not a function of heat accumulation. In our study, the peak temperature after each pulse decreased to baseline prior to each successive pulse. A temperature increase of 25 °C is possible with five consecutive 30 ms pulses of 18 J/cm² with a 9 s time interval. We show that in healthy, human-like skin, multi-pulse IPL can achieve selective vascular and perivascular damage with little thermal injury to the epidermis and dermis after proper cooling during treatment. The thermal damage of the vessels could have been enhanced due to an increased blood perfusion that is caused by the multiple pulses. The IPL system provides the clinician a treatment modality with more adjustable parameters to improve the clinical response of various skin types and vascular lesions. We theorize that port-wine stains should respond to multi-pulse IPL therapy in a similar fashion to our healthy, human skin model.