Development of the WeChat Public Account I Love Parasitology and its Preliminary Application in the Teaching of Human Parasitology.

OBJECTIVE: To better construct teaching resources, enhance real-time interaction and feedback between teachers and students in and out of class, and improve the teaching quality of parasitology, our team set up a WeChat public account I love Parasitology. METHODS: The data sources were mainly from original pictures and multimedia materials of different parasites collected and produced by our team, as well as related materials collected from traditional publications and digital media. With the instant interactive platform, course schedules and corresponding teaching contents were sent by push notifications, case-based learning was carried out, and 2-way communication between students and teachers was achieved. Teaching effectiveness was assessed using a self-evaluation questionnaire. RESULTS: A WeChat public account suitable for our daily teaching of parasitology was established. The second recursion and implementation of the learning resources allowed students to conduct in-depth reading and get unrestricted access to high-quality resources through the public account. In addition, all contents were in digital forms and made the original resources reborn, which would make up for our current and future shortage of physical teaching specimens. Moreover, the results from the questionnaire indicated that all these actions encouraged students to master theoretical knowledge, improved their abilities of case analysis and communication, and increased their knowledge of academic progress. CONCLUSION: Our WeChat public account can provide excellent learning materials for students and is a good supplement to the routine education of human parasitology.

MiR-429 regulates blood-spinal cord barrier permeability by targeting Krüppel-like factor 6.

The blood-spinal cord barrier (BSCB) is an effective, tightly-connected tissue that reduces secondary spinal cord injury (SCI) by decreasing blood cell infiltration, inflammation, and neuronal cell death during primary SCI. However, the methods and molecular mechanisms of BSCB openness remain elusive. In the present study, we found that microRNA429 (miR-429) plays a vital role in the opening of the blood-spinal cord. Inhibiting the expression of miR-429 (antagomiR-429) resulted in increased expression levels of the tight junction (TJ) proteins, ZO-1, occludin, and claudin-5, in the BSCB and reduced BSCB permeability. Moreover, overexpression of miR-429 (agomiR-429) had the opposite effect. Krüppel-like factor 6 (KLF6) is a transcription factor of the zinc-finger family. Using RT-qPCR and western blotting, we found that miR-429 can negatively regulate the expression of the KLF6. Co-transfection of KLF6 and miR-429 demonstrated that miR-429 negatively regulates KLF6 to mediate TJ protein expression and BSCB permeability. Based on these results, we suggest that KLF6 may be a downstream target of miR-429, mediating TJ protein expression to regulate the BSCB.

[Observation on mitosis of Trichomonas vaginalis cultivated in vitro using modified Giemsa staining].

The observation showed that the percentage of Trichomonas vaginalis trophozoites at the stages of interphase, binary fission and multiple fission was 66.5%, 24.1% and 9.4% respectively. Cells in binary fission could be classified as premitotic phase, prophase, metaphase, anaphase and telophase. 3 to 8 microcosms were seen in one trophozoite under multiple fission and the percentage of trophozoites with 3 and 4 microcosms occupied 69% and 24.5% respectively. Cells with abnormal morphs were also observed.

[Construction and expression of Schistosoma japonicum bivalent DNA vaccines and their efficiencies of protective immunity].

OBJECTIVE: To study the efficiency of protective immunity afforded by the constructed bivalent DNA vaccines of Schistosoma japonicum. METHODS: The plasmids pVIVO2-mcs-SjFABP-Sj23 and pVIVO2-mes-Sj23-SjFABP, co-expressed bivalent DNA vaccines, were constructed and identified. The presence of bivalent DNA vaccine in the mouse muscle cells was also tested by indirect immunofluorescent antibody tests (IFAT). 70 BALB/c mice were randomly divided into seven groups to be injected with normal saline, pVIVO2-mcs, pVIVO2-mcs-Sj23, pVIVO2-mcs-SjFABP, pVIVO2-mcs-Sj23-SjFABP, pVIVO2-mcs-SjFABP-Sj23 plasmid DNA, and a mixture of pVIVO2-mcs-Sj23-SjFABP plasmid DNA and amylose adjuvant, respectively. At day 45 after challenge the mice were sacrificed. The number of adult worms and hepatic eggs were counted. RESULT: Successful construction of co-expressed bivalent DNA vaccines were identified by restriction analysis and sequencing. It was confirmed by IFAT that the bivalent DNA vaccine was expressed in the plasma and on the surface of muscle cells from mouse. The worm reduction rate was 41.20%-53.85% and the egg reduction rate was 47.02%-53.83% bivalent DNA groups. Furthermore, the worm and egg reduction rates in pVIVO2-mcs-Sj23-SjFABP plasmid DNA and amylose adjuvant group were 68.89% and 84.04% respectively, significantly higher than single antigenic DNA vaccine and bivalent DNA vaccine (P < 0.05). CONCLUSION: The co-expressed bivalent DNA vaccines of Schistosoma japonicum can induce immune protection in mice. The protective immunity of amylose adjuvant group is higher than that of the univalent DNA and bivalent vaccines.

Enhanced transscleral lontophoretic transport with ion-exchange membrane.

PURPOSE: Transscleral iontophoresis has been recently re-examined for drug delivery to the back of the eye. In conventional iontophoresis, due to the relatively high electromobility of the endogenous competing ions (counterions) relative to that of the drug ion in the tissue barrier, the efficiency of iontophoretic drug delivery is generally low. The objective of the present study was to examine ion-exchange membrane-enhanced transscleral iontophoretic transport in which the ion-exchange membrane in series with the sclera can hinder the transport of the competing counterions and selectively allow the transport of the permeant across the sclera. METHODS: The physical properties of the Ionac ion-exchange membrane and excised rabbit sclera were determined in equilibrium uptake experiments and in passive and iontophoretic transport experiments with salicylate, tetraethylammonium, urea, and mannitol. Transscleral experiments with the ion-exchange membrane were conducted with salicylate and excised rabbit sclera in vitro. The contribution of electroosmosis to electrotransport during transscleral iontophoresis was assessed with urea and mannitol. RESULTS: The ion-exchange membrane is highly positively charged and has a small effective pore size. The sclera is relatively porous with a large effective pore size and low pore tortuosity. The sclera is also net negatively charged but this does not significantly affect the transport of small ions. A three-fold steady-state transscleral flux enhancement of salicylate was observed in ion-exchange membrane-enhanced iontophoresis over conventional transscleral iontophoresis without the membrane. Such enhancement was relatively independent of the applied electric current density and the thickness of the studied ion-exchange membrane assembly. Although the ion-exchange membrane altered transscleral electroosmosis, the contribution of electroosmosis to electrotransport was not significant. CONCLUSIONS: The present study has demonstrated the potential of ion-exchange membranes for enhancing iontophoretic transport and drug delivery.

[The adjuvant effect of IL-12 on protective immunity of Schistosoma japonicum fatty acid binding protein (Sj14FABP)].

OBJECTIVE: To study the immune protection of Schistosoma japonicum fatty acid binding protein (Sj14FABP) DNA vaccine enhanced by IL-12. METHODS: The recombinant plasmids pVIVO2-Sj14FABP and pVIVO2-IL12-Sj14FABP were constructed respectively, and were prepared on a large scale after identification. 48 male BALB/c mice were divided into 4 groups randomly. In group A, B, C, and D, each mouse was injected intramuscularly with 100 microl 0.9% NaCl, 100 microg pVIVO2, 100 microg pVIVO2-Sj14FABP and 100 microg pVIVO2-IL12-Sj14FABP respectively. 30 days after immunization each mouse was challenged with 40 +/- 2 cercariae of S. japonicum. On day 45 after challenge, all mice were sacrificed to count the number of recovered adult worms and the hepatic eggs. Sera from mice were used to detect IgG antibody. The production of IL-2, IL-4 and IFN-gamma in the supernatant of spleen cells was observed by means of sandwich ABC-ELISA. RESULTS: The recombinant plasmids pVIVO2-Sj14FABP and pVIVO2-IL12-Sj14FABP were constructed. The worm reduction rate in group C and D was 24.11% and 39.4%, as well as liver egg reduction rate of 27.2% and 32.8% respectively. The level of IL-2 and IFN-gamma in group D increased significantly, while IL-4 secretion decreased (P < 0.01). 30 days after immunization, no higher titer of IgG antibody was shown in all groups. Furthermore, no significant difference on the level of IgG was found among the groups (P > 0.05). CONCLUSION: Sj14FABP DNA vaccine induces partial protective immunity in BALB/c mice. IL-12 drives the immune response toward a Th1 direction, and enhances the protective immune effect of the vaccine.

[Protective immunity induced by multivalent DNA vaccine of Schistosoma japonicum Mr23 x 10(3) membrane antigen and IL-12 in mice].

OBJECTIVE: To develop multivalent DNA vaccine PV-IL12-Sj23 which co-expresses Sj23 and cytokine IL-12, and investigate its protective efficacy in BALB/c mice against challenge infection. METHODS: On the basis of the reconstructed plasmid PV-IL12-Sj23 and plasmid PV-IL12, blank plasmid PV and plasmid PV-Sj23 only expressing Sj23 were constructed. Fifty BALB/c male mice were divided into five groups, which were immunized intramuscularly with multivalent DNA vaccine PV-IL12-Sj23, plasmid PV-Sj23 expressing Sj23, plasmid PV-IL12 expressing cytokine IL-12, blank plasmid PV and saline, respectively. Each mouse was immunized with 100 microg DNA only once. All the mice were challenged with 40 cercariae at week 4, killed and perfused for collection of worms at week10. The number of recovered worms and eggs in the liver were counted. RESULTS: Blank plasmid PV and plasmid PV-Sj23 expressing Sj23 were successfully constructed. The worm reduction rate in PV-IL12-Sj23 group and PV-Sj23 group was 45.5% and 27.2% (P< 0.05) respectively. The number of eggs in liver tissue was reduced by 58.4% and 33.9% respectively. CONCLUSION: Multivalent DNA vaccine PV-IL12-Sj23 can induce protective immunity against Schistosoma japonicum in BALB/c mice significantly, with a better protective efficacy than the monovalent DNA vaccine PV-Sj23.

[Vaccination of mice with recombinant nucleic acid vaccine encoding the integral membrane protein Sj23 and cytokine IL-12 elicits specific immune responses against schistosoma japonica].

OBJECTIVE: To develop a Schistosoma japonica integral membrane protein Sm23 or Sj23 combined with murine IL-12 DNA-base vaccine against schistosomiasis. METHODS: Plasmids pVIVO2-Sj23 and pVIVO2-IL12-Sj23, expressing the integral membrane protein Sj23 of Schistosoma japonica and/or murine IL-12 were constructed. The plasmid pVIVO2-IL12-Sj23 was transfected into the human embryonic kidney cells of the line 293. RT-PCR was used to detect the expression of Sj23 mRNA in the 293 cells. Indirect immunofluorescence test was used to detect the expression of Sj23 protein. Fifty BALB/c mice were randomly divided into 5 equal groups to be injected with pVIVO2-IL12-Sj23 plasmid DNA, pVIVO2-Sj23 plasmid DNA, pVIVO2-IL12 plasmid DNA, pVIVO2 blank vector, and normal saline respectively into the quadriceps muscle of thigh. Four weeks after each mouse were infested with 40 +/- 2 cercariae of Schistosoma japonica. Six weeks after the infestation the mice were killed to calculate the load of schistosoma and the amount of eggs per gram (EPG) of liver so as to calculate the worm reduction rate and egg reduction rate after the vaccination. Before immunization, 4 weeks after immunization, and 6 weeks after immunization blood samples were collected from the caudal veins of mice. With soluble egg antigen (SEA) and adult worm antigen (AWA) ELISA was used to detect the serum IgG level. Western blotting was used to detect the serum specific anti-Sj23 IgG level. Six weeks after the cercaria challenge single splenocyte suspension was prepared. Splenocytes were cultured with SEA, and concanavalin A (ConA). ELISA was used to detect the levels of IL-4 and IFN-gamma in the supernatant. Flow cytometry was used to analyze the subgroups of splenocyte. RESULTS: + Forty-eight hours after the transfection, RT-PCR and indirect immunofluorescence test showed expression of Sj23 mRNA and protein in the HEK-293 cells. The worm reduction rate was 45.53% and the egg reduction was 58.35% in the pVIVO2-IL12-Sj23 group, significantly higher than those in the monovalent vaccine pVIVO2-Sj23 group (27.23% and 33.93% respectively, both P <0.05). ELISA and Western blotting analysis showed that the level of IgG specific for Sj23 significantly increased 4 weeks after vaccination in the pVIVO2-IL12-Sj23 and pVIVO2-Sj23 groups without significant difference between these 2 groups (P > 0.05). After stimulation of ConA and SEA the level of Th1 type cell factor IFN-gamma was higher and the level of the Th2 type cellular factor IL-4 was low in the supernatant of suspension of splenocytes of the pVIVO2-IL12-Sj23 group. FCM showed the percentages of CD4+ and CD8+ subgroups of the murine splenocytes of all experimental groups were all significantly lower than those of the normal mice (all P < 0.001 approximately 0.02). However, there was no significant difference in the CD4+/CD8+ ratio among the experimental groups (all CONCLUSION: pVIVO2-IL12-Sj23 is sufficient to elicit significant levels of protective immunity against Schistosoma japonica challenge infection. IL-12, a cytokine and a gene adjuvant, is able to induce Th1 responses and hence the protective immunity.

Influence of asymmetric donor-receiver ion concentration upon transscleral iontophoretic transport.

Recent in vitro and in vivo studies have suggested transscleral iontophoresis as a means for non-invasive drug delivery to the eye. However, there remains a lack of information of the iontophoretic transport behavior of the sclera. The objective of the present study was to investigate the effects of permeant concentration upon transscleral iontophoretic transport. Constant current direct current (DC) iontophoresis was conducted with rabbit sclera in vitro at permeant concentration ranging from 0.015 to 1.0 M in the donor chamber without background electrolyte at 0.4-4 mA (current density: 2-20 mA/cm2). PBS (0.15 M) was the receiver solution. Salicylate (SA) and tetraethylammonium (TEA) were the model ionic permeants, and mannitol was the neutral probe permeant. Conductivity experiments of SA and TEA solutions were performed to determine the effects of ion concentration upon SA and TEA electromobilities. Model simulations were carried out and compared with the experimental data. It was found that the fluxes of the ionic permeants increased linearly with the electric current but were relatively independent of their donor concentrations. Electric field-induced convective solvent flow (electroosmosis) in the sclera was observed to be from the anode to cathode, suggesting that the sclera is net negatively charge at neutral pH. For the studied permeants, electrophoresis was the main transport enhancing mechanism with electroosmosis as a secondary effect. No significant interaction between the permeants and sclera was observed that significantly altered electroosmosis in the membrane. Under the asymmetric donor and receiver conditions, the transference of the permeants could not be predicted by the concentrations of the ions in the donor and receiver chambers with the assumption of constant electric field in the membrane. The membrane ion concentrations were different from those in the chambers due to the requirement of charge neutrality in the membrane.

Effects of electrophoresis and electroosmosis during alternating current iontophoresis across human epidermal membrane.

Previous studies in our laboratory have demonstrated that skin electrical resistance can be controlled by an alternating current (AC) electric field. By maintaining constant skin resistance, AC iontophoresis has been shown to reduce the iontophoretic flux variability of neutral permeants. Recently, it was found that symmetric square-wave AC could enhance iontophoretic transport of both neutral and ionic permeants by means of electrophoresis and/or electroosmosis in a synthetic membrane system, and a model was presented to describe the experimental results. The objective of the present study was to assess the effects of AC voltage and frequency and direct current (DC) offset on the flux of neutral and ionic model permeants with human epidermal membrane (HEM). Experiments were conducted under two different conditions: constant AC voltage iontophoresis and iontophoresis using constant HEM resistance with DC offset voltage. The following are the main findings in these experiments. In the constant AC voltage study, when the permeability data were compared at the same HEM electrical resistance, it was demonstrated that AC even at high frequency (approximately 1 kHz) could enhance the transport of the ionic permeant (tetraethylammonium ion) across HEM, but no enhancement was observed for the neutral permeant (arabinose). For the ionic permeant flux enhancement, the higher the applied AC voltage, the greater the flux enhancement. There was little or no AC frequency dependence of the flux enhancement in the frequency range of 50-1000 Hz. In the constant HEM resistance study of AC with DC offset, approximately linear relationships were observed between flux enhancement and the DC offset voltage for both the neutral and ionic permeants, and these results were found to be consistent with predictions of the modified Nernst-Planck model for conventional constant voltage DC iontophoresis. When the DC offset voltage was increased, the AC component of the flux enhancement for the ionic permeant decreased, eventually appearing to contribute negligibly to the total flux enhancement at high DC offset voltages.

Immune responses against Schistosoma japonicum after vaccinating mice with a multivalent DNA vaccine encoding integrated membrane protein Sj23 and cytokine interleukin-12.

BACKGROUND: The vaccination of mice with DNA encoding single candidate antigens has failed to induce significant protection against Schistosoma japonicum (S. japonicum) challenge infections. In this study, we evaluated the feasibility of using a multivalent DNA vaccine which co-expressed S. japonicum integral membrane protein Sj23 and murine cytokine IL-12 to induce protective immune responses. METHODS: The plasmid pVIVO2-IL12-Sj23, a eukaryotic expression vector expressing Sj23 and murine IL-12 simultaneously, was constructed, identified, and tested for expression in vitro. Its ability to protect against S. japonicum challenge infections was analyzed according to worm reduction rate and egg reduction rate after vaccination of BALB/c mice. The serum levels of specific IgG antibody were determined by enzyme-linked-immuno sorbent assay (ELISA) and Western blot analysis. Using cultured spleen cells, IFN-gamma and IL-4 post-stimulation were quantified by ELISA. The phenotypes of splenocyte populations were analyzed by flow cytometry (FCM). RESULTS: The plasmid DNA pVIVO2-IL12-Sj23 was proven to express well in vitro by transient transfection of HEK-293 cells. Immunization resulted in a worm reduction rate of 45.53% and egg reduction rate of 58.35%. ELISA and Western blot analysis indicated that immunized mice generated specific IgG against Sj23. Spleen cells showed significant increases in IFN-gamma but decreases in IL-4. No significant differences in CD4+ and CD8+ subgroup ratios were observed after the challenges. CONCLUSIONS: The multivalent DNA vaccine pVIVO2-IL12-Sj23 is sufficient to elicit moderate but highly significant levels of protective immunity against challenge infections. Cytokine IL-12, as a gene adjuvant, was able to enhance the Th1 responses and, hence, the protective immunity.

Quantitative study of electrophoretic and electroosmotic enhancement during alternating current iontophoresis across synthetic membranes.

One of the primary safety and tolerability limitations of direct current iontophoresis is the potential for electrochemical burns associated with the necessary current densities and/or application times required for effective treatment. Alternating current (AC) transdermal iontophoresis has the potential to eliminate electrochemical burns that are frequently observed during direct current transdermal iontophoresis. Although it has been demonstrated that the intrinsic permeability of skin can be increased by applying low-to-moderate AC voltages, transdermal transport phenomena and enhancement under AC conditions have not been systematically studied and are not well understood. The aim of the present work was to study the fundamental transport mechanisms of square-wave AC iontophoresis using a synthetic membrane system. The model synthetic membrane used was a composite Nuclepore membrane. AC frequencies ranging from 20 to 1000 Hz and AC fields ranging from 0.25 to 0.5 V/membrane were investigated. A charged permeant, tetraethyl ammonium, and a neutral permeant, arabinose, were used. The transport studies showed that flux was enhanced by increasing the AC voltage and decreasing AC frequency. Two theoretical transport models were developed: one is a homogeneous membrane model; the other is a heterogeneous membrane model. Experimental transport data were compared with computer simulations based on these models. Excellent agreement between model predictions and experimental data was observed when the data were compared with the simulations from the heterogeneous membrane model.

Mechanistic studies of flux variability of neutral and ionic permeants during constant current dc iontophoresis with human epidermal membrane.

Although constant current iontophoresis is supposed to provide constant transdermal transport, significant flux variability and/or time-dependent flux drifts are observed during iontophoresis with human skin in vitro and human studies in vivo. The objectives of the present study were to determine (a) the causes of flux variability in constant current dc transdermal iontophoresis and (b) the relationships of flux variabilities among permeants of different physicochemical properties. Changes in the human epidermal membrane (HEM) effective pore size and/or electroosmosis during constant current dc iontophoresis were examined. Tetraethylammonium ion (TEA), urea, and mannitol were the model permeants. For the neutral permeants, the results in the present study showed a significant increase of fluxes with time in a given experiment and large HEM sample-to-sample variability. Although both effective pore size and pore charge density variations contributed to the time-dependent flux drifts observed in electroosmotic transport, the significant flux drifts observed were found to be primarily a result of the time-dependent increase in effective pore charge density. For the ionic permeant, the observed flux variability was smaller than that of the neutral permeants and was believed to be primarily due to effective pore size alteration in HEM during iontophoresis as suggested in a previous study. The different extents of flux variability observed between neutral and ionic permeants are consistent with the different iontophoretically enhanced transport mechanisms for the neutral and ionic permeants (i.e. electroosmosis and electrophoresis, respectively). The results of the present study also demonstrate that flux variability of two neutral permeants are inter-related, so the flux of one neutral permeant can be predicted if the permeability coefficient of the other neutral permeant is known.

In vitro and in vivo comparisons of constant resistance AC iontophoresis and DC iontophoresis.

A previous in vitro constant electrical resistance alternating current (AC) iontophoresis study with human epidermal membrane (HEM) and a model neutral permeant has shown less inter- and intra-sample variability in iontophoretic transport relative to conventional constant direct current (DC) iontophoresis. The objectives of the present study were to address the following questions. (1) Can the skin electrical resistance be maintained at a constant level by AC in humans in vivo? (2) Are the in vitro data with HEM representative of those in vivo? (3) Does constant skin resistance AC iontophoresis have less inter- and intra-sample variability than conventional constant current DC iontophoresis in vivo? (4) What are the electrical and the barrier properties of skin during iontophoresis in vivo? In the present study, in vitro HEM experiments were carried out with the constant resistance AC and the conventional constant current DC methods using mannitol and glucose as the neutral model permeants. In vivo human experiments were performed using glucose as the permeant with a constant skin resistance AC only protocol and two conventional constant current DC methods (continuous constant current DC and constant current DC with its polarity alternated every 10 min with a 3:7 on:off duty cycle). Constant current DC iontophoresis was conducted with commercial constant current DC devices, and constant resistance AC iontophoresis was carried out by reducing and maintaining the skin resistance at a constant target value with AC supplied from a function generator. This study shows that (1) skin electrical resistance can be maintained at a constant level during AC iontophoresis in vivo; (2) HEM in vitro and human skin in vivo demonstrate similar electrical and barrier properties, and these properties are consistent with our previous findings; (3) there is general qualitative and semi-quantitative agreement between the HEM data in vitro and human skin data in vivo; and (4) constant skin resistance AC iontophoresis generally provides less inter- and intra-subject variability than conventional constant current DC.

Investigation of properties of human epidermal membrane under constant conductance alternating current iontophoresis.

Previous studies in our laboratory have shown that enhanced, constant permeant fluxes across human skin can be achieved by applying an alternating current (AC) to maintain skin electrical conductance at a constant level. Relative to conventional direct current (DC) iontophoresis, for which current is maintained at a constant level, this newly developed constant conductance alternating current (CCAC) method achieves constant fluxes with less inter- and intra-sample variability. The present study focused upon further investigating the permeability properties of human skin during CCAC iontophoresis at a variety of target resistance/conductance values. A three-stage experimental protocol was used with flux measurements determined on 3 consecutive days. Stage I was an AC only protocol (symmetrical AC square-wave signal), stage II was an AC plus DC protocol (AC square-wave with DC offset voltage), and stage III was a repeat of stage I. During this three-stage protocol, the skin electrical resistance was maintained at a constant target value by manually adjusting the applied AC voltage. Radiolabeled mannitol and urea were model permeants in all experiments. Their fluxes were determined and used to characterize the permeability properties of human skin. The results from the present study established that: (i) the CCAC protocol made it possible to reduce HEM electrical resistance to different target levels as low as 0.8 kOmega cm(2) and maintain the specific resistance level throughout the flux experiment, (ii) permeant fluxes are proportional to skin electrical conductance, (iii) under the studied CCAC passive conditions, membrane pore size tends to increase as skin resistance decreases, and (iv) as the membrane breaks down, its pore sizes become larger.

Improvement on conventional constant current DC iontophoresis: a study using constant conductance AC iontophoresis.

The purpose of the present study was to compare conventional constant direct current (DC) transdermal iontophoresis with a new constant conductance alternating current (AC) iontophoresis method. The new method was developed with the intent of reducing flux drift during iontophoresis and minimizing skin-to-skin variability. The constant conductance AC iontophoresis studies involved three electrical components: (1) an initial applied potential used to decrease the human epidermal membrane (HEM) electrical resistance to a target level of either 1.5 or 3.0 k Omega cm(2), (2) an applied 50 Hz square-wave AC with a variable potential adjusted to maintain the HEM conductance at the target level during the transport study, and (3) a low voltage DC offset of 0 (passive), 0.25, or 0.40 V applied simultaneously with the AC to assist permeant transport. Current densities of 0.13 and 0.26 mA/cm(2) were chosen for the conventional constant current DC iontophoresis studies. Mannitol was used as the probe permeant for all studies. The constant current DC studies showed significant increases in mannitol flux with time during a given experiment and large skin-to-skin variability. Compared to the constant current DC experiments, the mannitol flux remained more constant during the constant conductance AC iontophoresis and skin-to-skin variability was significantly reduced. On a mechanistic level, changes in the transport properties during constant current DC iontophoresis indicate changes in the membrane parameters such as porosity, effective pore size, and/or pore surface charge density during the conventional method of iontophoresis. The results from the constant conductance AC iontophoresis transport studies imply that this method effectively maintains the membrane parameters that affect transport at a constant state this providing for a relatively constant permanent flux.

Human epidermal membrane constant conductance iontophoresis: alternating current to obtain reproducible enhanced permeation and reduced lag times of a nonionic polar permeant.

An experimental protocol, using an initial 1 min direct current (DC) applied potential of 4 V followed by alternating current (AC), was established to: (a) increase conductance and permeability and decrease lag time for human epidermal membrane (HEM) relative to unaltered HEM and; (b) maintain constant conductance and permeability during flux studies. The protocol allowed specific permeation parameters of the membrane to be characterized under electrically enhanced, constant flux conditions. The permeability, lag time, and effective membrane thickness were determined using a nonionic polar permeant, urea, while the enhanced conductance was maintained at a constant level with AC. A tortuous pore pathway model was employed to analyze the data. The AC protocol increased membrane permeability, and decreased lag time and effective membrane thickness relative to similar parameters obtained in previous studies from unaltered HEM. Lag times ranged from 32.0 to 105.5 min, and permeability coefficients calculated from steady state fluxes ranged from 1.68 to 6.03x10(-7) cm/s for HEM samples with electrical resistance values during transport of 2.3-8.0 kOmega x cm2. Effective membrane thicknesses were calculated to range from 0.34 to 0.61 cm during AC iontophoresis. Significant additional results were obtained when the protocol was applied for two consecutive runs using the same HEM sample, with time for the HEM sample to recover between runs. During the second run, the applied potential was adjusted to reproduce the conductance obtained on the first run. Under these conditions, the consecutive runs yielded essentially the same lag time, permeability and effective membrane thickness values. These results suggest that constant fluxes can be achieved by keeping HEM electrical conductance constant during AC iontophoresis.