Intrinsic immunogenicity of liposomes for tuberculosis vaccines: Effect of cationic lipid and cholesterol.

Tuberculosis (TB) is still among the deadliest infectious diseases, hence there is a pressing need for more effective TB vaccines. Cationic liposome subunit vaccines are excellent vaccine candidates offering effective protection with a better safety profile than live vaccines. In this study, we aim to explore intrinsic adjuvant properties of cationic liposomes to maximize immune activation while minimizing aspecific cytotoxicity. To achieve this, we developed a rational strategy to select liposomal formulation compositions and assessed their physicochemical and immunological properties in vitro models using human monocyte-derived dendritic cells (MDDCs). A broad selection of commercially available cationic compounds was tested to prepare liposomes containing Ag85B-ESAT6-Rv2034 (AER) fusion protein antigen. 1,2-Dioleoyl-sn­glycero-3-ethylphosphocholine (EPC)-based liposomes exhibited the most advantageous activation profile in MDDCs as assessed by cell surface activation markers, cellular uptake, antigen-specific T-cell activation, cytokine production, and cellular viability. The addition of cholesterol to 20 mol% improved the performance of the tested formulations compared to those without it; however, when its concentration was doubled there was no further benefit, resulting in reduced cell viability. This study provides new insights into the role of cationic lipids and cholesterol in liposomal subunit vaccines.

Intradermal Administration of Influenza Vaccine with Trehalose and Pullulan-Based Dissolving Microneedle Arrays.

Most influenza vaccines are administered via intramuscular injection which has several disadvantages that might jeopardize the compliance of vaccinees. Intradermal administration of dissolving-microneedle-arrays (dMNAs) could serve as minimal invasive alternative to needle injections. However, during the production process of dMNAs antigens are subjected to several stresses, which may reduce their potency. Moreover, the needles need to have sufficient mechanical strength to penetrate the skin and subsequently dissolve effectively to release the incorporated antigen. Here, we investigated whether blends of trehalose and pullulan are suitable for the production of stable dMNA fulfilling these criteria. Our results demonstrate that production of trehalose/pullulan-based dMNAs rendered microneedles that were sharp and stiff enough to pierce into ex vivo human skin and subsequently dissolve within 15 min. The mechanical properties of the dMNAs were maintained well even after four weeks of storage at temperatures up to 37°C. In addition, immunization of mice with influenza antigens via both freshly prepared dMNAs and dMNAs after storage (four weeks at 4°C or 37°C) resulted in antibody titers of similar magnitude as found in intramuscularly injected mice and partially protected mice from influenza virus infection. Altogether, our results demonstrate the potential of trehalose/pullulan-based dMNAs as alternative dosage form for influenza vaccination.

Diphtheria toxoid dissolving microneedle vaccination: Adjuvant screening and effect of repeated-fractional dose administration.

In this study the effect of repeated-fractional intradermal administration of diphtheria toxoid (DT) compared to a single administration in the presence or absence of adjuvants formulated in dissolving microneedles (dMNs) was investigated. Based on an adjuvant screening with a hollow microneedle (hMN) system, poly(I:C) and gibbsite, a nanoparticulate aluminum salt, were selected for further studies: they were co-encapsulated with DT in dMNs with either a full or fractional DT-adjuvant dose. Sharp dMNs were prepared regardless the composition and were capable to penetrate the skin, dissolve within 20 min and deposit the intended antigen-adjuvant dose, which remained in the skin for at least 5 h. Dermal immunization with hMN in repeated-fractional dosing (RFrD) resulted in a higher immune response than a single-full dose (SFD) administration. Vaccination by dMNs led overall to higher responses than hMN but did not show an enhanced response after RFrD compared to a SFD administration. Co-encapsulation of the adjuvant in dMNs did not increase the immune response further. Immunization by dMNs without adjuvant gave a comparable response to subcutaneously injected DT-AlPO4 in a 15 times higher dose of DT, as well as subcutaneous injected DT-poly(I:C) in a similar DT dose. Summarizing, adjuvant-free dMNs showed to be a promising delivery tool for vaccination performed in SFD administration.

Chemical Modifications of Gold Surfaces with Basic Groups and a Fluorescent Nanoparticle Adhesion Assay To Determine Their Surface p Ka.

For pharmaceutical, biological, and biomedical applications, the functionalization of gold surfaces with pH-sensitive groups has great potential. The aim of this work was to modify gold surfaces with pH-sensitive groups and to determine the p Ka of the modified gold surfaces using a fluorescent nanoparticle adhesion assay. To introduce pH-sensitive groups onto gold surfaces, we modified gold-coated silicon slides with four different bases: 4-mercaptopyridine (4-MP), 4-pyridylethylmercaptan (4-PEM), 4-aminothiophenol (4-ATP), and 2-mercaptoethylamine (2-MEA). To screen whether the modifications were successful, the binding of negatively charged fluorescently labeled nanoparticles to the positively charged surfaces was visualized by fluorescence microscopy and atomic force microscopy. Next, the p Ka of the modified surfaces was determined by quantifying the pH-dependent adhesion of the fluorescently labeled nanoparticles with fluorescence spectroscopy. Fluorescence microscopy showed that the gold surfaces were successfully modified with the four different basic molecules. Moreover, fluorescence spectroscopy revealed that fluorescently labeled negatively charged nanoparticles bound onto gold surfaces that were modified with one of the four bases in a pH-dependent manner. By quantifying the adsorption of negatively charged fluorescently labeled nanoparticles onto the functionalized gold surfaces and using the Henderson-Hasselbalch equation, the p Ka of these surfaces was determined to be 3.7 ± 0.1 (4-MP), 5.0 ± 0.1 (4-PEM), 5.4 ± 0.1 (4-ATP), and 7.4 ± 0.3 (2-MEA). We successfully functionalized gold surfaces with four different basic molecules, yielding modified surfaces with different p Ka values, as determined with a fluorescent nanoparticle adhesion assay.

New insight into phase behavior and permeability of skin lipid models based on sphingosine and phytosphingosine ceramides.

The intercellular lipid matrix of the stratum corneum (SC), which consist mainly of ceramides (CERs), free fatty acids and cholesterol, is fundamental to the skin barrier function. These lipids assemble into two lamellar phases, known as the long and short periodicity phases (LPP and SPP respectively). The LPP is unique in the SC and is considered important for the skin barrier function. Alterations in CER composition, as well as impaired skin barrier function, are commonly observed in diseased skin, yet the understanding of this relationship remains insufficient. In this study, we have investigated the influence of non-hydroxy and α-hydroxy sphingosine-based CERs and their phytosphingosine counterparts on the permeability and lipid organization of model membranes, which were adjusted in composition to enhance formation of the LPP. The permeability was compared by diffusion studies using ethyl-p-aminobenzoate as a model drug, and the lipid organization was characterized by X-ray diffraction and infrared spectroscopy. Both the sphingosine- and phytosphingosine-based CER models formed the LPP, while the latter exhibited a longer LPP repeat distance. The ethyl-p-aminobenzoate flux across the sphingosine-based CER models was higher when compared to the phytosphingosine counterparts, contrary to the fact that the α-hydroxy phytosphingosine-based CER model had the lowest chain packing density. The unanticipated low permeability of the α-hydroxy phytosphingosine-based model is probably associated with a stronger headgroup hydrogen bonding network. Our findings indicate that the increased level of sphingosine-based CERs at the expense of phytosphingosine-based CERs, as observed in the diseased skin, may contribute to the barrier function impairment.

Compromising human skin in vivo and ex vivo to study skin barrier repair.

Ex vivo regenerated stratum corneum (SC) after tape-stripping can be used as a model to study the barrier function of compromised skin. Yet, details about how close the regenerated SC model mimics the lipid properties (e.g. lipid composition and lipid ordering) of the in vivo situation are not known. Here, we examined using a comprehensive ceramide analysis whether human ex vivo regenerated SC showed similar lipid properties as human in vivo regenerated SC. Both in vivo and ex vivo regenerated SC had an altered ceramide subclass composition, with increased percentages of sphingosine-based subclass and decreased percentages of phytosphingosine-based subclass ceramides, a reduced mean ceramide chain length, and a higher percentage of unsaturated ceramides. Overall, regenerated SC ex vivo showed more pronounced but similar changes compared to the in vivo response. One of the purposes of these models is to use them to mimic compromised skin of inflammatory skin diseases. The altered lipid properties in regenerated SC were comparable to those observed in several inflammatory skin diseases, which makes them a valuable model for the barrier properties in inflammatory skin diseases.

Interactions of dipalmitoylphosphatidylcholine with ceramide-based mixtures.

The outermost layer of the skin, the stratum corneum (SC), acts as the natural physical barrier. The SC consists of corneocytes embedded in a crystalline lipid matrix consisting of ceramides, free fatty acids and cholesterol. Although phospholipids are frequently present in topical formulations, no detailed information is reported on the interactions between phospholipids and SC lipids. The aim of this study was to examine the interactions between a model phospholipid, dipalmitoylphosphatidylcholine (DPPC) and synthetic ceramide-based mixtures (referred to as SC lipids). (Perdeuterated) DPPC was mixed with SC lipids and the lipid organization and mixing properties were examined. The studies revealed that DPPC participates in the same lattice as SC lipids thereby enhancing a hexagonal packing. Even at a high DPPC level, no phase separated pure DPPC was observed. When a DPPC containing formulation is applied to the skin surface it must partition into the SC lipid matrix prior to any mixing with the SC lipids. To mimic this, DPPC was applied on top of a SC lipid membrane. DPPC applied in a liquid crystalline state was able to mix with the SC lipids and participated in the same lattice as the SC lipids. However, when DPPC was applied in a rippled gel-state very limited partitioning of DPPC into the SC lipid matrix occurred. Thus, when applied to the skin, liquid crystalline DPPC will have very different interactions with SC lipids than DPPC in a (rippled-)gel phase.

Dissolving Microneedle Patches for Dermal Vaccination.

The dermal route is an attractive route for vaccine delivery due to the easy skin accessibility and a dense network of immune cells in the skin. The development of microneedles is crucial to take advantage of the skin immunization and simultaneously to overcome problems related to vaccination by conventional needles (e.g. pain, needle-stick injuries or needle re-use). This review focuses on dissolving microneedles that after penetration into the skin dissolve releasing the encapsulated antigen. The microneedle patch fabrication techniques and their challenges are discussed as well as the microneedle characterization methods and antigen stability aspects. The immunogenicity of antigens formulated in dissolving microneedles are addressed. Finally, the early clinical development is discussed.

Stratum corneum lipid matrix: Location of acyl ceramide and cholesterol in the unit cell of the long periodicity phase.

The extracellular lipid matrix in the skin's outermost layer, the stratum corneum, is crucial for the skin barrier. The matrix is composed of ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) and involves two lamellar phases: the short periodicity phase (SPP) and the long periodicity phase (LPP). To understand the skin barrier thoroughly, information about the molecular arrangement in the unit cell of these lamellar phases is paramount. Previously we examined the molecular arrangement in the unit cell of the SPP. Furthermore X-ray and neutron diffraction revealed a trilayer arrangement of lipids within the unit cell of the LPP [D. Groen et al., Biophysical Journal, 97, 2242-2249, 2009]. In the present study, we used neutron diffraction to obtain more details about the location of lipid (sub)classes in the unit cell of the LPP. The diffraction pattern revealed at least 8 diffraction orders of the LPP with a repeating unit of 129.6±0.5Å. To determine the location of lipid sub(classes) in the unit cell, samples were examined with either only protiated lipids or selectively deuterated lipids. The diffraction data obtained by means of D2O/H2O contrast variation together with a gradual replacement of one particular CER, the acyl CER, by its partly deuterated counterpart, were used to construct the scattering length density profiles. The acyl chain of the acyl CER subclass is located at a position of ~21.4±0.2Å from the unit cell centre of the LPP. The position and orientation of CHOL in the LPP unit cell were determined using tail and head-group deuterated forms of the sterol. CHOL is located with its head-group positioned ~26±0.2Å from the unit cell centre. This allows the formation of a hydrogen bond with the ester group of the acyl CER located in close proximity. Based on the positions of the deuterated moieties of the acyl CER, CHOL and the previously determined location of two other lipid subclasses [E.H. Mojumdar et al., Biophysical Journal, 108, 2670-2679, 2015], a molecular model is proposed for the unit cell of the LPP. In this model CHOL is located in the two outer layers of the LPP, while CER EOS is linking the two outer layers with the central lipid layers. Finally the two other lipid subclasses are predominantly located in the central layer of the LPP.

Phase behavior of skin lipid mixtures: the effect of cholesterol on lipid organization.

The lipid matrix in the stratum corneum (SC), the upper layer of the skin, plays a critical role in the skin barrier. The matrix consists of ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs). In human SC, these lipids form two coexisting crystalline lamellar phases with periodicities of approximately 6 and 13 nm. In the studies reported here, we investigated the effect of CHOL on lipid organization in each of these lamellar phases separately. For this purpose, we used lipid model mixtures. Our studies revealed that CHOL is imperative for the formation of each of the lamellar phases. At low CHOL levels, the formation of the lamellar phases was dramatically changed: a minimum 0.2 CHOL level in the CER/CHOL/FFA (1 : 0.2 : 1) mixture is required for the formation of each of the lamellar phases. Furthermore, CHOL enhances the formation of the highly dense orthorhombic lateral packing. The gradual increment of CHOL increases the fraction of lipids forming the very dense orthorhombic lateral packing. Therefore, these studies demonstrate that CHOL is an indispensable component of the SC lipid matrix and is of fundamental importance for appropriate dense lipid organization and thus important for the skin barrier function.

Monitoring the penetration process of single microneedles with varying tip diameters.

Microneedles represent promising tools for delivery of drugs to the skin. However, before these microneedles can be used in clinical practice, it is essential to understand the process of skin penetration by these microneedles. The present study was designed to monitor both penetration depth and force of single solid microneedles with various tip diameters ranging from 5 to 37µm to provide insight into the penetration process into the skin of these sharp microneedles. To determine the microneedle penetration depth, single microneedles were inserted in human ex vivo skin while monitoring the surface of the skin. Simultaneously, the force on the microneedles was measured. The average penetration depth at 1.5mm displacement was similar for all tip diameters. However, the process of penetration depth was significantly different for the various microneedles. Microneedles with a tip diameter of 5µm were smoothly inserted into the skin, while the penetration depth of microneedles with a larger tip diameter suddenly increased after initial superficial penetration. In addition, the force at insertion (defined as the force at a sudden decrease in measured force) linearly increased with tip diameter ranging from 20 to 167mN. The force drop at insertion was associated with a measured penetration depth of approximately 160µm for all tip diameters, suggesting that the drop in force was due to the penetration of a deeper skin layer. This study showed that sharp microneedles are essential to insert microneedles in a well-controlled way to a desired depth.

The role of ceramide chain length distribution on the barrier properties of the skin lipid membranes.

The skin barrier function is provided by the stratum corneum (SC). The lipids in the SC are composed of three lipid classes: ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) which form two crystalline lamellar structures. In the present study, we investigate the effect of CER chain length distribution on the barrier properties of model lipid membranes mimicking the lipid composition and organization of SC. The membranes were prepared with either isolated pig CERs (PCERs) or synthetic CERs. While PCERs have a wide chain length distribution, the synthetic CERs are quite uniform in chain length. The barrier properties were examined by means of permeation studies using hydrocortisone as a model drug. Our studies revealed a reduced barrier in lipid membranes prepared with PCERs compared to synthetic CERs. Additional studies revealed that a wider chain length distribution of PCERs results in an enhanced hexagonal packing and increased conformational disordering of the lipid tails compared to synthetic CERs, while the lamellar phases did not change. This demonstrates that the chain length distribution affects the lipid barrier by reducing the lipid ordering and density within the lipid lamellae. In subsequent studies, the effect of increased levels of FFAs or CERs with a long acyl chain in the PCERs membranes was also studied. These changes in lipid composition enhanced the level of orthorhombic packing, reduced the conformational disordering and increased the barrier of the lipid membranes. In conclusion, the CER chain length distribution is an important key factor for maintaining a proper barrier.

Lipid to protein ratio plays an important role in the skin barrier function in patients with atopic eczema.

BACKGROUND: The barrier function of the skin is primarily provided by the stratum corneum (SC), the outermost layer of the skin. Skin barrier impairment is thought to be a primary factor in the pathogenesis of atopic eczema (AE). Filaggrin is an epidermal barrier protein and common mutations in the filaggrin gene strongly predispose for AE. However, the role of filaggrin mutations in the decreased skin barrier in AE is not fully understood. It was recently shown that changes in SC lipid composition and organization play a role in the reduced skin barrier in AE. OBJECTIVES: To determine whether the lipid/protein ratio and the total dry SC mass per surface area are related to the skin barrier function of controls and patients with AE. METHODS: A case-control study was performed to compare nonlesional and lesional skin of AE with skin of controls. The dry SC mass was determined by tape-stripping and Squamescan(™) . The ratio between lipid and protein bands in the Raman spectrum was used to determine the lipid/protein ratio. Skin barrier function was assessed by transepidermal water loss. RESULTS: The results show that the dry SC mass per skin area is altered only in lesional SC of patients with AE compared with control subjects. The observed reduction in the lipid/protein ratio in SC of patients with AE was more pronounced, both in lesional and nonlesional SC and correlated strongly with the skin barrier function and disease severity. CONCLUSIONS: The lipid/protein ratio plays a role in the reduced skin barrier function in AE.

The important role of stratum corneum lipids for the cutaneous barrier function.

The skin protects the body from unwanted influences from the environment as well as excessive water loss. The barrier function of the skin is located in the stratum corneum (SC). The SC consists of corneocytes embedded in a lipid matrix. This lipid matrix is crucial for the lipid skin barrier function. This paper provides an overview of the reported SC lipid composition and organization mainly focusing on healthy and diseased human skin. In addition, an overview is provided on the data describing the relation between lipid modulations and the impaired skin barrier function. Finally, the use of in vitro lipid models for a better understanding of the relation between the lipid composition, lipid organization and skin lipid barrier is discussed. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.

Localization of cholesterol and fatty acid in a model lipid membrane: a neutron diffraction approach.

The intercellular lipid matrix of the skin's stratum corneum serves to protect the body against desiccation and simultaneously limits the passage of drugs and other xenobiotics into the body. The matrix is made up of ceramides, free fatty acids, and cholesterol, which are organized as two coexisting crystalline lamellar phases. In studies reported here, we sought to use the technique of neutron diffraction, together with the device of isotopic (H/D) substitution, to determine the molecular architecture of the lamellar phase having a repeat distance of 53.9 ± 0.3 Å. Using hydrogenous samples as well as samples incorporating perdeuterated (C24:0) fatty acids and selectively deuterated cholesterol, the diffraction data obtained were used to construct neutron scattering length density profiles. By this means, the locations within the unit cell were determined for the cholesterol and fatty acids. The cholesterol headgroup was found to lie slightly inward from the unit cell boundary and the tail of the molecule located 6.2 ± 0.2 Å from the unit cell center. The fatty acid headgroups were located at the unit cell boundary with their acyl chains straddling the unit cell center. Based on these results, a molecular model is proposed for the arrangement of the lipids within the unit cell.

Electron diffraction study of lipids in non-lesional stratum corneum of atopic eczema patients.

Skin barrier impairment is thought to be an important factor in the pathogenesis of atopic eczema (AE). The skin barrier is located in the stratum corneum (SC), consisting of corneocytes embedded in lipids. Ceramides, cholesterol and free fatty acids are the major lipid classes and are crucial for the skin barrier function, but their role in relation to AE is indistinct. Filaggrin is an epidermal barrier protein and common mutations in the filaggrin gene strongly predispose for AE. However, there is no strong evidence that filaggrin mutations are related to the reduced skin barrier in AE. In this study, electron diffraction is used in order to study the lipid organization of control SC and non-lesional SC of AE patients in vivo. An increased presence of the hexagonal lipid organization was observed in non-lesional SC of AE patients, indicating a less dense lipid organization. These changes correlate with a reduced skin barrier function as measured with transepidermal water loss but do not correlate with the presence of filaggrin mutations. These results are indicative for the importance of the lipid organization for a proper skin barrier function.

Human skin equivalents are an excellent tool to study the effect of moisturizers on the water distribution in the stratum corneum.

In this study, the mode of action of moisturizers on the level of water in the stratum corneum was studied using cryo-scanning electron microscopy. As model for dry skin, we used human skin equivalents (HSEs) generated at 93% or 60% relative humidity (RH). During the generation of the HSEs, the moisturizers were applied during a period of maximal 2 weeks. In HSEs generated under normal culture conditions (93% RH), application of 10% glycerol or 5% urea formulations resulted in increased water levels. Whereas the 5% urea formulations resulted mainly in the formation of intercellular water domains, after 10% glycerol both swelling of corneocytes and formation of intercellular water domains were noticed. A reduction in RH to 60% during treatment reduced the stratum corneum water levels drastically. Treatment with the non-occlusive lipophilic moisturizer isopropyl isostearate resulted in increased water level in the central part of the stratum corneum compared with the untreated control. Our results show that HSEs can be used as a model to study the water distribution.

Disposition of ceramide in model lipid membranes determined by neutron diffraction.

The lipid matrix present in the uppermost layer of the skin, the stratum corneum, plays a crucial role in the skin barrier function. The lipids are organized into two lamellar phases. To gain more insight into the molecular organization of one of these lamellar phases, we performed neutron diffraction studies. In the diffraction pattern, five diffraction orders were observed attributed to a lamellar phase with a repeat distance of 5.4 nm. Using contrast variation, the scattering length density profile could be calculated showing a typical bilayer arrangement. To obtain information on the arrangement of ceramides in the unit cell, a mixture that included a partly deuterated ceramide was also examined. The scattering length density profile of the 5.4-nm phase containing this deuterated ceramide demonstrated a symmetric arrangement of the ceramides with interdigitating acyl chains in the center of the unit cell.

The in vitro and in vivo evaluation of new synthesized prodrugs of 5-OH-DPAT for iontophoretic delivery.

The feasibility of transdermal iontophoretic transport of 4 novel ester prodrugs of 5-OH-DPAT (glycine-, proline-, valine- and beta-alanine-5-OH-DPAT) was investigated in vitro and in vivo. Based on the chemical stability of the prodrugs, the best candidates were selected for in vitro transport studies across human skin. The pharmacokinetics and pharmacodynamic effects of the prodrug with highest transport efficiency, were investigated in a rat model. The in vitro transport, plasma profile and pharmacological response were analyzed with compartmental modeling. Valine- and beta-alanine-5-OH-DPAT were acceptably stable in the donor phase and showed a 4-fold and 14-fold increase in solubility compared to 5-OH-DPAT. Compared to 5-OH-DPAT, valine- and beta-alanine-5-OH-DPAT were transported less and more efficiently across human skin, respectively. Despite a higher in vitro transport, lower plasma concentration was observed following 1.5h current application (250 microAcm(2)) of beta-alanine-S-5-OH-DPAT in comparison to S-5-OH-DPAT. However the prodrug showed higher plasma concentrations post-iontophoresis, explained by a delayed release due to hydrolysis and skin depot formation. This resulted in a pharmacological effect with the same maximum as 5-OH-DPAT, but the effect lasted for a longer time. The current findings suggest that beta-alanine-5-OH-DPAT is a promising prodrug, with a good balance between stability, transport efficiency and enzymatic conversion.

Comparing different salt forms of rotigotine to improve transdermal iontophoretic delivery.

The transdermal delivery of a new salt form of the dopamine agonist rotigotine, rotigotine.H(3)PO(4) is presented and compared to rotigotine.HCl. A comparison was made on the level of solubility, passive and iontophoretic delivery. Different aspects of the delivery were investigated: delivery efficiency, maximum flux, donor pH, electro-osmotic contribution and transport number. Changing the salt form from rotigotine.HCl to rotigotine.H(3)PO(4) increases significantly the solubility and rules out the influence of NaCl on the solubility by the absence of the common-ion effect. At low donor concentration, no difference in transdermal delivery was observed between the salt forms. Due to an increase in the maximum solubility of rotigotine.H(3)PO(4), a 170% increase in maximum flux, compared to rotigotine.HCl, was achieved. A balance between solubility and delivery efficiency can be obtained by choosing the correct donor pH between 5 and 6. A slight increase in electro-osmotic contribution and transport number was observed. Using the parameters, determined by modeling the in vitro transport, in vivo simulations revealed that with iontophoresis therapeutic levels can be achieved with a rapid onset time and be maintained in a controlled manner by adjusting the current density.