Biomedical subjects

Daniel Blankschtein

Publications and source records attributed to Daniel Blankschtein.

At least 19 recordsLinked to original sources

PubMed2006

New methodology to determine the rate-limiting adsorption kinetics mechanism from experimental dynamic surface tension data.

We present a new methodology to determine the rate-limiting adsorption kinetics mechanism (diffusion-controlled vs mixed diffusion-barrier controlled), including deducing the kinetics parameters (the diffusion coefficient, D, and the energy-barrier parameter, beta), from the experimental short-time dynamic surface tension (DST) data. The new methodology has the following advantages over the existing procedure used to analyze the experimental DST data: (a) it does not require using a model for the equilibrium adsorption isotherm, and (b) it only requires using the experimental short-time DST data measured at two initial surfactant bulk solution concentrations. We apply the new methodology to analyze the experimental short-time DST data of the following alkyl poly(ethylene oxide), CiEj, nonionic surfactants: C12E4, C12E6, C12E8, and C10E8 measured using the pendant-bubble apparatus. We find that for C12E4 and C12E6, the effect of the energy barrier on the overall rate of surfactant adsorption can be neglected for surfactant bulk solution concentrations below their respective critical micelle concentrations (CMCs), and therefore, that the rate-limiting adsorption kinetics mechanism for C12E4 and C12E6 is diffusion-controlled at any of their premicellar surfactant bulk solution concentrations. On the other hand, for C12E8 and C10E8, we find that their respective CMC values are large enough to observe a significant effect of the energy barrier on the overall rate of surfactant adsorption. In other words, for C12E8 and C10E8, the rate-limiting adsorption kinetics mechanism shifts from diffusion-controlled to mixed diffusion-barrier controlled as their premicellar surfactant bulk solution concentrations increase. We test the new methodology by predicting the short-time DST profiles at other initial surfactant bulk solution concentrations, and then comparing the predicted DST profiles with those measured experimentally. Very good agreement is obtained for the four CiEj nonionic surfactants considered. We also compare the results of implementing the new methodology with those of implementing the existing procedure, and conclude that using a model for the equilibrium adsorption isotherm can lead not only to different values of D and beta, but it can also lead to a completely different determination of the rate-limiting adsorption kinetics mechanism. Since the new methodology proposed here does not require using a model for the equilibrium adsorption isotherm, we conclude that it should provide a more reliable determination of the rate-limiting adsorption kinetics mechanism, including the deduced kinetics parameters, D and beta.

Journal Article↗

PubMed2006

Molecular-thermodynamic theory of micellization of pH-sensitive surfactants.

A predictive, molecular-thermodynamic theory is developed to model the micellization of pH-sensitive surfactants. The theory combines a molecular-thermodynamic description of micellization in binary surfactant mixtures with the protonation equilibrium of the surfactant monomers. The thermodynamic component of the theory models the pH-mediated equilibrium between micelles, surfactant monomers, and counterions. These counterions may originate from the surfactant or from added salt, acid, or base. The molecular component of the theory models the various contributions to the free energy of micellization, which corresponds to the free-energy change associated with forming a mixed micelle from the protonated and deprotonated forms of the surfactant and from the bound counterions. The free energy of micellization includes hydrophobic, interfacial, packing, steric, electrostatic, and entropic contributions, which are all calculated molecularly. The theory also requires knowledge of the surfactant molecular structure and the solution conditions, including the temperature and the amount of any added salt, acid, or base. To account for the pH sensitivity of the surfactant, the theory requires knowledge of the surfactant monomer equilibrium deprotonation constant (pK1), which may be obtained from experimental titration data obtained below the critical micelle concentration (cmc). The theory can be utilized to predict the equilibrium micelle and solution properties, including the cmc, the micelle composition, the micelle shape and aggregation number, the solution pH, and the micelle deprotonation equilibrium constant (pKm). Theoretical predictions of the cmc, the micelle aggregation number, and the pKm compare favorably with the available experimental data for alkyldimethylamine oxide surfactants. This class of pH-sensitive surfactants exhibits a form of self-synergy, which has previously been attributed to hydrogen-bond formation at the micelle interface. Instead, we show that much of the observed synergy is related to the electrostatic contribution to the free energy of micellization. Although we do not explicitly include hydrogen bonding in the molecular model of micellization, we briefly discuss how it may be incorporated and its anticipated effect on the predicted micellization behavior.

Journal Article↗

PubMed2006

Affinity-tagged green fluorescent protein (GFP) extraction from a clarified E. coli cell lysate using a two-phase aqueous micellar system.

Green fluorescent protein (GFP) has been proposed as an ideal choice for a protein-based biological indicator for use in the validation of decontamination or disinfection treatments. In this article, we present a potentially scalable and cost-effective way to purify recombinant GFP, produced by fermentation in Escherichia coli, by affinity-enhanced extraction in a two-phase aqueous micellar system. Affinity-enhanced partitioning, which improves the specificity and yield of the target protein by specific bioaffinity interactions, has been demonstrated. A novel affinity tag, family 9 carbohydrate-binding module (CBM9) is fused to GFP, and the resulting fusion protein is affinity-extracted in a decyl beta-D-glucopyranoside (C10G1) two-phase aqueous micellar system. In this system, C10G1 acts as phase forming and as affinity surfactant. We will further demonstrate the implementation of this concept to attain partial recovery of affinity-tagged GFP from a clarified E. coli cell lysate, including the simultaneous removal of other contaminating proteins. The cell lysate was partitioned at three levels of dilution (5x, 10x, and 40x). Irrespective of the dilution level, CBM9-GFP was found to partition preferentially to the micelle-rich phase, with the same partition coefficient value as that found in the absence of the cell lysate. The host cell proteins from the cell lysate were found to partition preferentially to the micelle-poor phase, where they experience less excluded-volume interactions. The demonstration of proof-of-principle of the direct affinity-enhanced extraction of CBM9-GFP from the cell lysate represents an important first step towards developing a cost-effective separation method for GFP, and more generally, for other proteins of interest.

Affinity Labels↗

PubMed2006

Complementary use of simulations and molecular-thermodynamic theory to model micellization.

Molecular-thermodynamic descriptions of micellization in aqueous media can be utilized to model the self-assembly of surfactants possessing relatively simple chemical structures, where it is possible to identify a priori what equilibrium position they will adopt in the resulting micellar aggregate. For such chemical structures, the portion of the surfactant molecule that is expected to be exposed to water upon aggregate self-assembly can be identified and used as an input to the molecular-thermodynamic description. Unfortunately, for many surfactants possessing more complex chemical structures, it is not clear a priori how they will orient themselves within a micellar aggregate. In this paper, we present a computational approach to identify what portions of a surfactant molecule are hydrated in a micellar environment through the use of molecular dynamics simulations of such molecules at an oil/water interface (modeling the micelle core/water interface). The local environment of each surfactant segment is determined by counting the number of contacts of each segment with the water and oil molecules. After identifying the hydrated and the unhydrated segments of the surfactant molecule, molecular-thermodynamic modeling can be performed to predict: (i) the free-energy change associated with forming a micellar aggregate, (ii) the critical micelle concentration (CMC), and (iii) the optimal shape and size of the micellar aggregate. The computer simulation results were found to be sensitive to the atomic charge parameters utilized during the simulation runs. Two different methods of assigning atomic charges were tested, and the computer simulation and molecular-thermodynamic modeling results obtained using both sets of atomic charges are presented and compared. The combined computer simulation/molecular-thermodynamic modeling approach presented here is validated first by implementing it in the case of anionic (sodium dodecyl sulfate, SDS), cationic (cetyltrimethylammonium bromide, CTAB), zwitterionic (dodecylphosphocholine, DPC), and nonionic (dodecyl poly(ethylene oxide), C12E8) surfactants possessing relatively simple chemical structures and verifying that good predictions of CMCs and micelle aggregation numbers are obtained. In the case of C12E8, the challenges and limitations associated with simulating a single, polymeric E8 moiety at the oil/water interface to model its behavior at the micelle/water interface are discussed. Subsequently, the combined modeling approach is implemented in the case of the anionic surfactant 3-hydroxy sulfonate (AOS) and of the nonionic surfactant decanoyl-n-methylglucamide (MEGA-10), which possess significantly more complex chemical structures. The good predictions obtained for these two surfactants indicate that the combined computer simulation/molecular-thermodynamic modeling approach presented here extends the range of applicability of molecular-thermodynamic theory to allow modeling of the micellization behavior of surfactants possessing more complex chemical structures.

Micelles↗

PubMed2006

Experimental and theoretical investigation of the micellar-assisted solubilization of ibuprofen in aqueous media.

Surfactants can be used to increase the solubility of poorly soluble drugs in water and to increase drug bioavailability. In this article, the aqueous solubilization of the nonsteroidal, antiinflammatory drug ibuprofen is studied experimentally and theoretically in micellar solutions of anionic (sodium dodecyl sulfate, SDS), cationic (dodecyltrimethylammonium bromide, DTAB), and nonionic (dodecyl octa(ethylene oxide), C12E8) surfactants possessing the same hydrocarbon "tail" length but differing in their hydrophilic headgroups. We find that, for these three surfactants, the aqueous solubility of ibuprofen increases linearly with increasing surfactant concentration. In particular, we observed a 16-fold increase in the solubility of ibuprofen relative to that in the aqueous buffer upon the addition of 80 mM DTAB and 80 mM C12E8 but only a 5.5-fold solubility increase upon the addition of 80 mM SDS. The highest value of the molar solubilization capacity (chi) was obtained for DTAB (chi = 0.97), followed by C12E8 (chi = 0.72) and finally by SDS (chi = 0.23). A recently developed computer simulation/molecular-thermodynamic modeling approach was extended to predict theoretically the solubilization behavior of the three ibuprofen/surfactant mixtures considered. In this modeling approach, molecular-dynamics (MD) simulations were used to identify which portions of ibuprofen are exposed to water (hydrated) in a micellar environment by simulating a single ibuprofen molecule at an oil/water interface (modeling the micelle core/water interface). On the basis of this input, molecular-thermodynamic modeling was then implemented to predict (i) the micellar composition as a function of surfactant concentration, (ii) the aqueous solubility of ibuprofen as a function of surfactant concentration, and (iii) the molar solubilization capacity (chi). Our theoretical results on the solubility of ibuprofen in aqueous SDS and C12E8 surfactant solutions are in good agreement with the experimental data. The ibuprofen solubility in aqueous DTAB solutions was somewhat overpredicted because of challenges associated with accurately modeling the strong electrostatic interactions between the anionic ibuprofen and the cationic DTAB. Our results indicate that computer simulations of ibuprofen at a flat oil/water interface can be used to obtain accurate information about the hydrated and the unhydrated portions of ibuprofen in a micellar environment. This information can then be used as input to a molecular-thermodynamic model of self-assembly to successfully predict the aqueous solubilization behavior of ibuprofen in the three surfactant systems studied.

Ibuprofen↗

PubMed2005

Modeling counterion binding in ionic-nonionic and ionic-zwitterionic binary surfactant mixtures.

A predictive molecular-thermodynamic theory is developed to model the effect of counterion binding on micellar solution properties of binary surfactant mixtures of ionic and nonionic (or zwitterionic) surfactants. The theory combines a molecular-thermodynamic description of micellization in binary surfactant mixtures with a recently developed model of counterion binding to single-component ionic surfactant micelles. The thermodynamic component of the theory models the equilibrium between the surfactant monomers, the counterions, and the mixed micelles. The molecular component of the theory models the various contributions to the free-energy change associated with forming a mixed micelle from ionic surfactants, nonionic (or zwitterionic) surfactants, and bound counterions (referred to as the free energy of mixed micellization). Specifically, the various molecular contributions to the free energy of mixed micellization model the underlying physics associated with the assembly of, and the interactions between, the surfactant polar heads, the surfactant nonpolar tails, and the bound counterions. Utilizing known structural characteristics of the surfactants and the counterions, along with the solution conditions, the free energy of mixed micellization is minimized to predict various optimal micelle characteristics, including the degree of counterion binding, the micelle composition, and the micelle shape and size. These predicted optimal micelle characteristics are then used to predict the critical micelle concentration (cmc) and the average micelle aggregation number. Our predictions of the degree of counterion binding, the cmc, and the average micelle aggregation number show good agreement with available experimental results from the literature for several binary surfactant mixtures. In addition, the theory is used to shed light on the relationship between the micelle composition, counterion binding and ion condensation, and the micelle shape transition.

Journal Article↗

PubMed2005

Short-time behavior of mixed diffusion-barrier controlled adsorption.

This paper focuses on the short-time adsorption kinetics of nonionic surfactants onto water/air surfaces, analyzed in the context of the mixed diffusion-barrier controlled adsorption modeling framework. Specifically, we reconcile the apparent contradiction between theoretical prediction and experimental observations on the adsorption kinetics mechanism at short times: while the mixed diffusion-barrier controlled model predicts a barrier-controlled adsorption, as well as the impossibility of a diffusion-controlled adsorption at asymptotic short times, the short-time experimental dynamic surface tension (DST) behavior of many nonionic surfactants has been interpreted to result from diffusion-controlled adsorption at asymptotic short times. This is because the short-time experimental DST of these surfactants displays a t variation, which is considered as a fingerprint for the existence of diffusion-controlled adsorption, based on the short-time asymptotic behavior of the diffusion-controlled adsorption model. As a result of this interpretation, the fundamental physical nature of the energy barrier has been proposed to be associated with high surfactant surface concentrations. In this paper, we derive a new nonasymptotic short-time formalism of the mixed diffusion-barrier controlled model to describe surfactant adsorption onto a spherical pendant-bubble surface, including determining the ranges of time and surfactant surface concentration values where the short-time formalism is applicable. Based on this formalism, we find that one can expect to observe an apparent t variation of the DST at short times even for the mixed diffusion-barrier controlled adsorption model. We analyze the consequence of this finding by re-evaluating the existing notions of the energy barrier. We conclude that the energy barrier is associated with the adsorption of a single surfactant molecule onto a clean surface.

Journal Article↗

PubMed2005

Affinity-enhanced protein partitioning in decyl beta-D-glucopyranoside two-phase aqueous micellar systems.

Liquid-liquid extraction in two-phase aqueous complex-fluid systems has been proposed as a scalable, versatile, and cost-effective purification method for the downstream processing of biotechnological products. In the case of two-phase aqueous micellar systems, careful choices of the phase-forming surfactants or surfactant mixtures allow these systems to separate biomolecules based on size, hydrophobicity, charge, or specific affinity. In this article, we investigate the affinity-enhanced partitioning of a model affinity-tagged protei--green fluorescent protein fused to a family 9 carbohydrate-binding module (CBM9-GFP)--in a two-phase aqueous micellar system generated from the nonionic surfactant n-decyl beta-D-glucopyranoside (C10G1), which acts simultaneously as the phase-former and the affinity ligand. In this simple system, CBM9-GFP was extracted preferentially into the micelle-rich phase, despite the opposing tendency of the steric, excluded-volume interactions operating between the protein and the micelles. We obtained more than a sixfold increase (from 0.47 to 3.1) in the protein partition coefficient (Kp), as compared to a control case where the affinity interactions were "turned off" by the addition of a competitive inhibitor (glucose). It was demonstrated conclusively that the observed increase in Kp can be attributed to the specific affinity between the CBM9 domain and the affinity surfactant C10G1, suggesting that the method can be generally applied to any CBM9-tagged protein. To rationalize the observed phenomenon of affinity-enhanced partitioning in two-phase aqueous micellar systems, we formulated a theoretical framework to model the protein partition coefficient. The modeling approach accounts for both the excluded-volume interactions and the affinity interactions between the protein and the surfactants, and considers the contributions from the monomeric and the micellar surfactants separately. The model was shown to be consistent with the experimental data, as well as with our current understanding of the CBM9 domain.

Chromatography, Affinity↗

PubMed2005

Prediction of conformational characteristics and micellar solution properties of fluorocarbon surfactants.

A molecular-thermodynamic theory is developed to model the micellization of fluorocarbon surfactants in aqueous solutions, by combining a molecular model that evaluates the free energy of micellization of fluorocarbon surfactant micelles with a previously developed thermodynamic framework describing the free energy of the micellar solution. In the molecular model of micellization developed, a single-chain mean-field theory is combined with an appropriate rotational isomeric state model of fluorocarbon chains to describe the packing of the fluorocarbon surfactant tails inside the micelle core. Utilizing this single-chain mean-field theory, the packing free energies of fluorocarbon surfactants are evaluated and compared with those of their hydrocarbon analogues. We find that the greater rigidity of the fluorocarbon chain promotes its packing in micellar aggregates of low curvatures, such as bilayers. In addition, the mean-field approach is utilized to predict the average conformational characteristics (specifically, the bond order parameters) of fluorocarbon and hydrocarbon surfactant tails within the micelle core, and the predictions are found to agree well with the available experimental results. The electrostatic effects in fluorocarbon ionic surfactant micelles are modeled by allowing for counterion binding onto the charged micelle surface, which accounts explicitly for the effect of the counterion type on the micellar solution properties. In addition, a theoretical formulation is developed to evaluate the free energy of micellization and the size distribution of finite disklike micelles, which often form in the case of fluorocarbon surfactants. We find that, compared to their hydrocarbon analogues, fluorocarbon surfactants exhibit a greater tendency to form cylindrical or disklike micelles, as a result of their larger molecular volume as well as due to the greater conformational rigidity of the fluorocarbon tails. The molecular-thermodynamic theory developed is then applied to several ionic fluorocarbon surfactant-electrolyte systems, including perfluoroalkanoates and perfluorosulfonates with added LiCl or NH(4)Cl, and various micellar solution properties, including critical micelle concentrations (cmc's), optimal micelle shapes, and average micelle aggregation numbers, are predicted. The predicted micellar solution properties agree reasonably well with the available experimental results.

Journal Article↗

PubMed2004

Protein partitioning driven by excluded-volume interactions in an aqueous nonionic micellar-gel system.

The separation and purification of biomolecules in aqueous media driven by excluded-volume interactions is a well-established concept. In this article we propose a new separations method, based on excluded-volume principles, consisting of an aqueous micellar-gel system (AMGS). Specifically, an outer aqueous phase containing cylindrically shaped micelles of the nonionic surfactant n-decyl tetra (ethylene oxide) (C10E4) is physically separated from an inner aqueous phase defined by the interior volume of gel beads, from which the micelles are completely excluded because of their shape and size. In the AMGS, the concentration of the micelles outside the gel beads is sufficiently high that the volume excluded to a biomolecule in the solution external to the gel beads is much larger than that within the gel beads. Accordingly, when biomolecules are introduced into the AMGS, they partition preferentially into the gel-bead phase, according to their sizes, as a result of the greater effect of the excluded-volume interactions with the C10E4 micelles present in the aqueous phase outside the gel beads. The new AMGS is more versatile and adaptable than the conventional two-phase aqueous C10E4 micellar system because the micelle volume fraction is independent of the temperature and because the effects of entrainment are eliminated. After demonstrating the experimental feasibility of creating the new AMGS, the three proteins myoglobin, ovalbumin, and BSA-FITC, and the enzyme G6PD, were partitioned in the AMGS and their partitioning behavior was found to follow the experimental excluded-volume trends dictated by the interactions of the biomolecules with the C10E4 micelles. Specifically, the measured partition coefficients of the four biomolecules into the micellar phase were found to be less than unity and to decrease with increasing biomolecule size. A theoretical description of the partitioning behavior of the biomolecules in the new AMGS was formulated, based on excluded-volume considerations, and the predicted biomolecule partition coefficients were found to compare favorably with those measured for the four biomolecules studied.

Chemical Fractionation↗

PubMed2004

Experimental demonstration of the existence of highly permeable localized transport regions in low-frequency sonophoresis.

Recent advances in low-frequency sonophoresis have focused on the existence of hypothesized localized transport regions (LTRs). However, there has been no actual experimental demonstration that the hypothesized LTRs are, in fact, localized regions of high permeability. Through a series of low-frequency sonophoresis experiments conducted with full-thickness pig skin, in the presence of the surfactant sodium lauryl sulfate (SLS), in which we have separately measured the transport of calcein through the LTRs, which have areas ranging from 10 to 40 mm(2), and the surrounding regions of the skin (the non-LTRs) by means of a novel masking technique, we demonstrate that the calcein permeability through the LTRs is approximately 80-fold higher than the calcein permeability through the non-LTRs, suggesting that the LTRs are structurally perturbed to a greater extent than the non-LTRs from the exposure to the ultrasound/SLS system. In addition, we propose basic models to predict the total skin transdermal permeability from the transdermal permeabilities of the LTRs and the non-LTRs, and then compare the predictions to the experimental data obtained from the masking experiments. We also demonstrate that both the LTRs and the non-LTRs exhibit significant decreases in skin electrical resistivity relative to untreated skin ( approximately 5000-fold and approximately 170-fold, respectively), suggesting the existence of two levels of significant skin structural perturbation due to ultrasound exposure in the presence of SLS. Finally, an analysis of the porosity/tortuosity ratio (epsilon/tau) values suggests that trans-cellular transdermal transport pathways are present within the highly permeable, and highly structurally perturbed, LTRs.

Animals↗

PubMed2003

Glucose-6-phosphate dehydrogenase partitioning in two-phase aqueous mixed (nonionic/cationic) micellar systems.

The enzyme glucose-6-phosphate dehydrogenase (G6PD) plays an important role in maintaining the level of NADPH and in producing pentose phosphates for nucleotide biosynthesis. It is also of great value as an analytical reagent, being used in various quantitative assays. In searching for new strategies to purify this enzyme, the partitioning of G6PD in two-phase aqueous mixed (nonionic/cationic) micellar systems was investigated both experimentally and theoretically. Our results indicate that the use of a two-phase aqueous mixed micellar system composed of the nonionic surfactant C(10)E(4) (n-decyl tetra(ethylene oxide)) and the cationic surfactant C(n)TAB (alkyltrimethylammonium bromide, n = 8, 10, or 12) can improve significantly the partitioning behavior of G6PD relative to that obtained in the two-phase aqueous C(10)E(4) micellar system. This improvement can be attributed to electrostatic attractions between the positively charged mixed (nonionic/cationic) micelles and the net negatively charged enzyme G6PD, resulting in the preferential partitioning of G6PD to the top, mixed micelle-rich phase of the two-phase aqueous mixed micellar systems. The effect of varying the cationic surfactant tail length (n = 8, 10, and 12) on the denaturation and partitioning behavior of G6PD in the C(10)E(4) /C(n)TAB/buffer system was investigated. It was found that C(8)TAB is the least denaturing to G6PD, followed by C(10)TAB and C(12)TAB. However, the C(10)E(4)/C(12)TAB/buffer system generated stronger electrostatic attractions with the net negatively charged enzyme G6PD than the C(10)E(4)/C(10)TAB/buffer and the C(10)E(4)/C(8)TAB/buffer systems, when using the same amount of cationic surfactant. Overall, the two-phase aqueous mixed (C(10)E(4)/C(10)TAB) micellar system yielded the highest G6PD partition coefficient of 7.7, with a G6PD yield in the top phase of 71%, providing the optimal balance between the denaturing effect and the electrostatic attractions for the three cationic surfactants examined. A recently developed theoretical framework to predict protein partition coefficients in two-phase aqueous mixed (nonionic/ionic) micellar systems was implemented, and the theoretically predicted G6PD partition coefficients were found to be in reasonable quantitative agreement with the experimentally measured ones.

Animals↗

PubMed2003

Evaluation of fluorescent probe surface intensities as an indicator of transdermal permeant distributions using wide-area two-photon fluorescence microscopy.

The application of high-speed two-photon fluorescence microscopy (HTPM) to examine transdermal transport processes has enabled the noninvasive visualization of permeant spatial distributions over a larger, more clinically relevant wide area of the skin. Earlier studies demonstrated that the transdermal fluorescent probe distribution over a 2 x 2 mm skin area was well represented by a significantly reduced sampling of the 400 microscale skin sites (100 x 100 microm) constituting the wide area. In the present study, the 400 microscale skin sites are considered individually, and the site-to-site variability in permeant distributions is used as a model to reflect the range in experimentally measured skin permeabilities resulting from the inherent stratum corneum structural heterogeneity. The correlation established between the permeant surface intensity and the corresponding permeant intensity gradient at each skin site provides an indication of the potential for screening transdermal permeant distributions solely based on the evaluation of microscale permeant surface intensities. The strong linear correlation between the intensity gradient and the surface intensity for the hydrophilic model permeant, sulforhodamine B, demonstrated that surface intensities provide a robust indicator of the corresponding transdermal probe distributions at the microscale. For the hydrophobic model permeant, rhodamine B hexyl ester, however, weak correlations were observed between these two parameters. This result suggests that the stratum corneum microscale surface intensity does not validly capture the corresponding intensity gradients for the entire range of skin permeabilities typically encountered as a result of the inherent stratum corneum heterogeneity.

Administration, Cutaneous↗

PubMed2003

Visualization of oleic acid-induced transdermal diffusion pathways using two-photon fluorescence microscopy.

In a novel application of dual-channel high-speed two-photon fluorescence microscopy, the skin autofluores-cence and the transdermal fluorescent model drug spatial distributions were imaged simultaneously over precisely the same spatial coordinates. The dual channels enable the detection of the fluorescence emission wavelengths characteristic of the endogenous (intrinsic) skin fluorophores, as well as of the rhodamine-based model drug intensity emission at a different wavelength range of the fluorescence emission spectrum. These fluorescent model drugs delineate the oleic acid induced changes in permeant diffusion with respect to the skin structural features over the 0.3 mm by 0.3 mm skin area imaged per skin sample. The dual-channel high-speed two-photon fluorescence microscopy studies presented here provide evidence for the existence of intracorneocyte diffusion in addition to the commonly cited lipid multilamellar transdermal pathway. The image quantification analysis methodology introduced in this paper reveals that intracorneocyte diffusion exists for the hydrophobic (rhodamine B hexyl ester) and for the hydrophilic (sulforhodamine B) model drugs, in the absence of oleic acid chemical enhancer action. The mechanism of oleic acid chemical enhancer action, however, depends on the model drug physicochemical properties, where the oleic acid induces hydrophobic model drug localization to the lipid multilamellar region, while increasing the hydrophilic model drug lipid to corneocyte partitioning.

Biological Transport↗

PubMed2002

Separating lysozyme from bacteriophage P22 in two-phase aqueous micellar systems.

This communication demonstrates that two-phase aqueous mixed (nonionic/ionic) micellar systems have the potential for improving the separation of proteins from viruses. Specifically, two separation experiments were performed to show that the addition of the anionic surfactant sodium dodecyl sulfate (SDS) to the two-phase aqueous nonionic n-decyl tetra(ethylene oxide) (C(10)E(4)) micellar system increases the yield of a model net positively charged protein, lysozyme, in the micelle-rich phase from 75 to 95%, while still maintaining approximately the same yield of a model net negatively charged virus, bacteriophage P22, in the micelle-poor phase (97% vs. 98%).

Bacteriophage P22↗

PubMed2002

Understanding viral partitioning in two-phase aqueous nonionic micellar systems: 1. Role of attractive interactions between viruses and micelles.

The partitioning behavior of viruses in the two-phase aqueous nonionic n-decyl tetra(ethylene oxide) (C10E4) micellar system cannot be fully explained by considering solely the repulsive, steric, excluded-volume interactions that operate between the viruses and the nonionic C10E4 micelles. Specifically, an excluded-volume theory developed recently by our group is not able to quantitatively predict the observed viral partition coefficients, even though this theory is capable of providing reasonable quantitative predictions of protein partition coefficients. To shed light on the discrepancy between the theoretically predicted and the experimentally measured viral partition coefficients, a central assumption underlying the excluded-volume theory that the viruses and the C10E4 micelles interact solely through repulsive, excluded-volume interactions was challenged in this study. In particular, utilizing bacteriophage P22 as a model virus, a competitive inhibition test and a partitioning study of the capsids of bacteriophage P22 were conducted. Based on the results of these two experimental studies, it was concluded that any attractive interactions between the tailspikes of bacteriophage P22 and the C10E4 micelles are negligible. Another experimental study was carried out wherein the partition coefficients of the model viruses, bacteriophages P22 and T4, were measured at various temperatures, and compared with those previously obtained for bacteriophage phiX174. This comparison also indicated that possible attractive, electromagnetic-induced interactions between the bacteriophage particles and the C10E4 micelles cannot be invoked to rationalize the observed discrepancy between the theoretically predicted and the experimentally measured viral partition coefficients.

Bacteriophage P22↗

PubMed2002

Understanding viral partitioning in two-phase aqueous nonionic micellar systems: 2. Effect of entrained micelle-poor domains.

Unlike the partitioning behavior of hydrophilic, water-soluble proteins, the partitioning behavior of viruses in the two-phase aqueous nonionic n-decyl tetra(ethylene oxide) (C10E4) micellar system cannot be fully explained using the excluded-volume theory developed recently by our group. A central assumption underlying the excluded-volume theory--that macroscopic phase separation equilibrium is attained--was therefore challenged experimentally and theoretically. Photographs of the two-phase aqueous C10E4 micellar system were taken for different volume ratios to demonstrate that the entrainment of micelle-poor (virus-rich) domains in the macroscopic, top, micelle-rich phase decreases with a decrease in the volume ratio. Partitioning experiments were then conducted with the model virus bacteriophage P22 and the model protein cytochrome c at different operating temperatures for different volume ratios. For bacteriophage P22, the measured viral partition coefficient at each temperature decreased by about an order of magnitude when the volume ratio was decreased from 10 to 0.1, which clearly indicated that entrainment is an important factor influencing viral partitioning. For cytochrome c, the measured protein partition coefficient did not change, which demonstrated that this entrainment effect negligibly influences protein partitioning. A new theoretical description of partitioning was also developed that combines the excluded-volume theory with this entrainment effect. In this theory, one fitted parameter--the volume fraction of entrained micelle-poor domains in the macroscopic, top, micelle-rich phase--is used to account for the entrainment. To fit this parameter, only a single partitioning experiment is required for a given volume ratio, irrespectively of the partitioning solute. The new theoretical description of partitioning yielded very good quantitative predictions of the viral partition coefficients. Accordingly, it can be concluded that the primary mechanisms governing viral partitioning in the two-phase aqueous C10E4 micellar system are the entrainment of micelle-poor (virus-rich) domains in the macroscopic, top, micelle-rich phase and the excluded-volume interactions that operate between the viruses and the micelles.

Bacteriophage P22↗

PubMed2002

Effects of low-frequency ultrasound on the transdermal permeation of mannitol: comparative studies with in vivo and in vitro skin.

The in vivo and the in vitro correlation of the effects of low-frequency ultrasound (low-frequency sonophoresis, LFS) on the percutaneous penetration of mannitol, a model hydrophilic permeant, was investigated using three in vitro skin models (including full-thickness and split-thickness pig skin, and heat-stripped human cadaver skin) and in vivo pig as the animal model. The central objective of this article was to identify the relevant in vitro skin models and ultrasound conditions that may be used in in vitro LFS studies to predict the effects of LFS in vivo on the transdermal delivery of hydrophilic permeants. In this article, by conducting comparative studies of the in vivo pig skin and of the three in vitro skin models under two LFS protocols (a constant ultrasound energy dose protocol, and a constant skin electrical resistance protocol), we demonstrated that: (1) under a constant ultrasound energy dose protocol (protocol A, 5 min LFS), no good correlation was observed between the in vivo skin and the in vitro skin models in terms of the measured skin permeabilities to mannitol. Moreover, the effects of LFS on the barrier functions of the in vivo pig skin, as measured by the enhancement ratio of the skin permeation rate of mannitol and by the reduction of the skin electrical resistance, are much more pronounced than those observed with the excised skin models in vitro; (2) under a constant skin electrical resistance protocol (protocol B) of LFS, a good correlation was found between the skin permeability to mannitol measured using the three in vitro skin models and that of the in vivo pig skin. This result indicates that by utilizing the skin electrical resistance as a quick indicator of the skin permeabilization state due to LFS, the three in vitro skin models can be utilized to predict the transport rate of mannitol across the in vivo skin during LFS; (3) by applying a recently developed skin porous-pathway theory, we demonstrated that within the range of LFS conditions examined, the three in vitro skin models exhibit similar transport properties to mannitol and similar skin effective pore radius values, and hence, represent equivalent skin models for the in vitro LFS studies in the case of hydrophilic permeants; (4) histological studies revealed that the LFS protocol that was shown to be efficacious in enhancing the skin penetration rate of mannitol across the in vivo pig skin, and was also utilized for the in vivo/in vitro skin comparative studies, is safe for the living skin; and (5) through measuring the skin concentration of mannitol in the presence and in the absence of the LFS treatment, we found that the LFS-induced flux enhancement outweighs the enhancement of the skin concentration of mannitol during the LFS studies both in vivo and in vitro. This result suggests that LFS represents a good method of enhancing the systemic absorption of hydrophilic permeants, while it does not significantly alter the vehicle-to-skin partition coefficient for the same class of permeants.

Administration, Cutaneous↗