Photochemically Aided Arteriovenous Fistula Creation to Accelerate Fistula Maturation.

Rates of arteriovenous fistula maturation failure are still high, especially when suboptimal size veins are used. During successful maturation, the vein undergoes lumen dilatation and medial thickening, adapting to the increased hemodynamic forces. The vascular extracellular matrix plays an important role in regulating these adaptive changes and may be a target for promoting fistula maturation. In this study, we tested whether a device-enabled photochemical treatment of the vein prior to fistula creation facilitates maturation. Sheep cephalic veins were treated using a balloon catheter coated by a photoactivatable molecule (10-8-10 Dimer) and carrying an internal light fiber. As a result of the photochemical reaction, new covalent bonds were created during light activation among oxidizable amino acids of the vein wall matrix proteins. The treated vein lumen diameter and media area became significantly larger than the contralateral control fistula vein at 1 week (p = 0.035 and p = 0.034, respectively). There was also a higher percentage of proliferating smooth muscle cells in the treated veins than in the control veins (p = 0.029), without noticeable intimal hyperplasia. To prepare for the clinical testing of this treatment, we performed balloon over-dilatation of isolated human veins and found that veins can tolerate up to 66% overstretch without notable histological damage.

Creating a Natural Vascular Scaffold by Photochemical Treatment of the Extracellular Matrix for Vascular Applications.

The development of bioscaffolds for cardiovascular medical applications, such as peripheral artery disease (PAD), remains to be a challenge for tissue engineering. PAD is an increasingly common and serious cardiovascular illness characterized by progressive atherosclerotic stenosis, resulting in decreased blood perfusion to the lower extremities. Percutaneous transluminal angioplasty and stent placement are routinely performed on these patients with suboptimal outcomes. Natural Vascular Scaffolding (NVS) is a novel treatment in the development for PAD, which offers an alternative to stenting by building on the natural structural constituents in the extracellular matrix (ECM) of the blood vessel wall. During NVS treatment, blood vessels are exposed to a photoactivatable small molecule (10-8-10 Dimer) delivered locally to the vessel wall via an angioplasty balloon. When activated with 450 nm wavelength light, this therapy induces the formation of covalent protein-protein crosslinks of the ECM proteins by a photochemical mechanism, creating a natural scaffold. This therapy has the potential to reduce the need for stent placement by maintaining a larger diameter post-angioplasty and minimizing elastic recoil. Experiments were conducted to elucidate the mechanism of action of NVS, including the molecular mechanism of light activation and the impact of NVS on the ECM.

Photosensitized Oxidative Dimerization at Tyrosine by a Water-Soluble 4-Amino-1,8-naphthalimide.

The oxidation of proteins generates reactive amino acid (AA) residue intermediates, leading to protein modification and cross-linking. Aerobic studies with peptides and photosensitizers allow for the controlled generation of reactive oxygen species (ROS) and reactive AA residue intermediates, providing mechanistic insights as to how natural protein modifications form. Such studies have inspired the development of abiotic methods for protein modification and crosslinking, including applications of biomedical importance. Dityrosine linkages derived from oxidation at tyrosine (Tyr) residues represent one of the more well-understood oxidation-induced modifications. Here we demonstrate an aerobic, visible light-dependent oxidation reaction of Tyr-containing substrates promoted by a water-soluble 4-amino-1,8-naphthalimide-based photosensitizer. The developed procedure converts Tyr-containing substrates into o,o'-Tyr-Tyr linked dimers. The regioselectively formed o,o'-Tyr-Tyr linkage is consistent with dimeric standards prepared using a known enzymatic method. A crossover study with two peptides provides a statistical mixture of three distinct o,o'-Tyr-Tyr linked dimers, supporting a mechanism that involves Tyr residue oxidation followed by intermolecular combination.

Solvent-Dependent Photophysics and Reactivity of Monomeric and Dimeric 4-Amino-1,8-Naphthalimides.

The solvent-dependent photophysics of two 4-amino-substituted 1,8-naphthalene imides (AIs) were studied using fluorescence spectroscopy and laser flash photolysis. The compounds were functionalized with water-soluble 2,2'(ethylenedioxy) diethylamine groups, yielding a monomer (AI3) and a dimer (AI4). The radiative and nonradiative singlet-state deactivation processes of AI3 and AI4 were quantified in 10 solvents and at different pH values. The fluorescence quantum yield for the AI4 dimer in water was more than 100× lower than in other solvents, or for the monomeric AI3. The enhanced nonradiative decay of aqueous solutions of dimeric AI4 is accompanied by biexponential decay kinetics, suggesting equilibration with a dark excited state. An oxygen-quenchable triplet state (T1) of AI3 was produced upon 416 nm excitation in both water and n-octanol. In water, the T1 state evolved into a long-lived transient that was unreactive toward oxygen or several electron donors. This species was not observed in n-octanol. The transient observed upon 416 nm excitation of AI4 in water was extremely weak. However, production of T1 in both AI3 and AI4 was evidenced by the photoinduced electron transfer to methyl viologen, albeit in low quantum yield (0.0503 and 0.00778 for AI3 and AI4, respectively). The photophysics and reactivity are consistent with the production of an intramolecular charge transfer (ICT) state that is stabilized in water. Significantly, this stabilization enhances nonradiative decay pathways, particularly in the AI4 dimer. The results indicate that the photochemistry of these compounds can be environmentally mediated, switching from radical- to triplet-initiated processes.

Photochemical Modification of the Extracellular Matrix to Alter the Vascular Remodeling Process.

Therapeutic interventions for vascular diseases aim at achieving long-term patency by controlling vascular remodeling. The extracellular matrix (ECM) of the vessel wall plays a crucial role in regulating this process. This study introduces a novel photochemical treatment known as Natural Vascular Scaffolding, utilizing a 4-amino substituted 1,8-naphthimide (10-8-10 Dimer) and 450 nm light. This treatment induces structural changes in the ECM by forming covalent bonds between amino acids in ECM fibers without harming vascular cell survival, as evidenced by our results. To further investigate the mechanism of this treatment, porcine carotid artery segments were exposed to 10-8-10 Dimer and light activation. Subsequent experiments subjected these segments to enzymatic degradation through elastase or collagenase treatment and were analyzed using digital image analysis software (MIPAR) after histological processing. The results demonstrated significant preservation of collagen and elastin structures in the photochemically treated vascular wall, compared to controls. This suggests that photochemical treatment can effectively modulate vascular remodeling by enhancing the resistance of the ECM scaffold to degradation. This approach shows promise in scenarios where vascular segments experience significant hemodynamic fluctuations as it reinforces vascular wall integrity and preserves lumen patency. This can be valuable in treating veins prior to fistula creation and grafting or managing arterial aneurysm expansion.

Silicone elastomer uptake method for determination of free 1-alkyl-2-pyrrolidone concentration in micelle and hydroxypropyl-beta-cyclodextrin systems used in skin transport studies.

Previous investigations in our laboratory demonstrated how the polar head group and alkyl chain of amphiphilic chemical skin permeation enhancers contribute to enhancer potency. In those studies enhancers with n-alkyl chain lengths of eight or less were investigated. In order to investigate enhancers with longer n-alkyl chain lengths, enhancer-solubilizing agents should be considered. Corticosterone (CS) flux enhancement along the lipoidal pathway of hairless mouse skin (HMS) was determined with the enhancers 1-hexyl- (HP), 1-octyl- (OP), 1-decyl- (DP), and 1-dodecyl-2-pyrrolidone (DoP) solubilized in 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol-2000] (DSPE) micelles or in hydroxypropyl-beta-cyclodextrin (HPbetaCD). The free CS, HP, OP, DP, and DoP aqueous concentrations in the DSPE micelle and HPbetaCD systems were determined using a partitioning method. Comparisons of the enhancer potencies based on the free concentration of the enhancers revealed a nearly semi-logarithmic linear relationship between enhancer potency and the carbon number of the alkyl chain length with a slope of approximately 0.55. The observed n-alkyl chain length dependency in the aqueous phase is consistent with the hydrophobic effect. This study shows that longer chain enhancers may be studied by employing a solubilizing system, and free enhancer concentration in these systems can be determined with the aid of the silicone elastomer uptake method.

Model analysis of flux enhancement across hairless mouse skin induced by chemical permeation enhancers.

Previous permeant partitioning studies with hairless mouse skin (HMS) in the presence of several chemical skin permeation enhancers have revealed that, when such enhancers induce significant skin permeability coefficient enhancement, it is accompanied by significant enhancement in the equilibrium uptake (partitioning) of the permeant into the intercellular lipid component of the stratum corneum (SC). Particularly, it was found that the 1-alkyl-2-pyrrolidones and the 1-alkyl-2-azacycloheptanones, at aqueous solution concentrations that gave skin permeation enhancement (E) of 10 for corticosterone (CS, the permeant), enhanced the equilibrium uptake of beta-estradiol (E2beta, a surrogate permeant) from the aqueous phase into the intercellular lipids of HMS SC by a factor of 5-7. This finding raised the question of whether this uptake enhancement induced by the permeation enhancer under equilibrium conditions would be essentially the same as that determined kinetically from time-dependent permeation experiments utilizing appropriate SC membrane models and Fick's laws of diffusion to treat the data. HMS transport experiments were conducted with CS as the permeant and 1-octyl-2-pyrrolidone (OP) and 1-hexyl-2-azacyloheptanone (HAZ) as the enhancers. In treating the experimental data, a one-layer skin transport model (SC only) and a two-layer model (SC layer and the epidermis/dermis layer) were both investigated. Both the partition coefficient enhancement (E(K)) and the diffusion coefficient enhancement (E(D)) were deduced from the data treatment. The results showed that when the total transport enhancement of CS was around 11, E(K) was in the range of 6-8 and E(D) was in the range of 1.5-1.9 using both the one-layer and the two-layer models. This E(K) value was found to be in good agreement with the E2beta partition enhancement obtained directly under equilibrium conditions in previous studies. This indicates that (a) the rate-limiting domain for the transport of the lipophilic permeants across HMS and the HMS SC intercellular lipid domain probed in the equilibrium partitioning experiments are essentially the same, and (b) the total flux enhancement (E) of lipophilic permeants across HMS was driven mainly by enhancing the partitioning of the permeant into the rate-limiting domain (E(K)) and secondarily by enhancing the diffusion coefficients (E(D)) of the permeant in the domain. Comparison of the one-layer and two-layer skin model results revealed that non-steady-state transport of lipophilic compounds across HMS was better described by the two-layer model because the dermis/viable epidermis played a significant role in lipophilic permeant binding.

Mechanistic study of alkyl azacycloheptanones as skin permeation enhancers by permeation and partition experiments with hairless mouse skin.

In previous studies (Yoneto et al., 1995. J Pharm Sci 84:312-317; Kim et al., 1992. Int J Pharm 80:17-31; and Warner et al., 2001. J Pharm Sci 90:1143-53), the transport enhancing effects of four homologous series of enhancers-the n-alkanols, 1-alkyl-2-pyrrolidones, 1,2-alkanediols, and N,N-dimethylalkanamides - on the transport of steroidal permeants across hairless mouse skin (HMS) were investigated. Isoenhancement concentrations are defined as the aqueous concentrations for which different enhancers induce the same extent of permeant transport enhancement, E, for the lipoidal pathway of the stratum corneum (SC). Our studies have shown that the E = 10 isoenhancement concentrations of these four homologous series were nearly the same when compared at the same n-alkyl group chain length and therefore that the contribution of the polar head group toward the enhancer potency was found to be essentially constant. In the present study, we have determined the isoenhancement concentrations (E = 10) for the 1-alkyl-2-azacycloheptanone series [1-butyl-2-azacycloheptanone (BAZ), 1-hexyl-2-azacycloheptanone (HAZ), and 1-octyl-2-azacycloheptanone (OAZ)] and compared the results with those of the previously studied four homologous series. We have found that the E = 10 isoenhancement concentrations (aqueous phase concentrations) of the 1-alkyl-2-azacycloheptanones (Azs) are around 10 times lower than those for the previously studied four homologous series when compared at the same alkyl group chain length. This indicates an approximately 10 times higher potency of Azs. This finding was a point of interest because the polar group of Azs is similar to that of 1-alkyl-2-pyrrolidones (Aps). To further probe the nature of the mechanism of action of the Azs and Aps and to better understand the lower E = 10 isoenhancement concentrations found with the Azs, it was decided (a) to determine the equilibrium partitioning (uptake) of the Azs and the Aps from the aqueous phase into the HMS SC at E = 10, and (b) to determine the equilibrium partitioning (uptake) of a surrogate permeant, estradiol (E2beta), into the SC in the absence of and in the presence of Azs and Aps at E = 10. The following were the outcomes from the two partitioning studies. Firstly, at the E = 10 isoenhancement concentrations, the extent of partitioning (uptake) of the Azs and Aps into the intercellular lipids of the HMS SC was found to be approximately the same, even though the E = 10 isoenhancement concentrations (aqueous phase concentrations) of the Aps were around 10 times greater than those of the Azs. We interpret this to mean (whereas the potencies of the Azs are around ten times greater than those of the Aps when related to their aqueous concentrations) that the potencies of the two enhancer series are about the same when expressed in terms of their concentrations in the intercellular lipid phase of the SC. Another outcome of the partitioning studies has been the finding that the extent of partitioning into the intercellular lipids of the SC at E = 10 isoenhancement conditions for both the Azs and Aps is essentially independent of the n-alkyl chain length (from butyl to octyl). A third result from these experiments has been that the partitioning of E2beta (the surrogate permeant) into the HMS SC under E = 10 isoenhancement concentration conditions is approximately the same with the Aps and Azs as enhancers. For both the Aps and Azs, the E2beta SC partitioning enhancement was found to be in the range of 5-6 at E = 10. This comparable partitioning enhancement for E2beta in the presence of Aps and Azs at E = 10 suggests that the same mechanism was involved and that these enhancers act, in part but to a significant extent, by inducing a higher partitioning tendency of the permeant into the transport rate-limiting lipoidal domains of the SC. (c) 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:297-310, 2003

Mechanistic study of chemical skin permeation enhancers with different polar and lipophilic functional groups.

In a previous study, the enhancement effects on the transport of a steroidal permeant along the hairless mouse skin (HMS) stratum corneum (SC) lipoidal pathway were investigated for two homologous series of chemical enhancers: the 1-alkyl-2-pyrrolidones and the 1-alkyl-2-azacycloheptanones. The objective of the present study was to extend this investigation to a broader range of enhancers in order that generalizations with regard to the mechanistic aspects of enhancer function might be established. Specific questions to be addressed included: (a) what is the nature of the microenvironment of the enhancer site of action? (b) what is the extent of the equilibrium uptake of the enhancer from its E = 10 aqueous enhancer solution (the aqueous concentration for which the enhancer induces a tenfold transport enhancement) into the HMS SC intercellular lipid "phase"? and (c) are the microenvironment of the enhancer site of action and that for the equilibrium enhancer uptake at E = 10 relatively independent of the molecular characteristics of the enhancers (as suggested by the earlier study)? Enhancers selected for this study included: a wide range of polar head group size and polarity; n-alkyl group chain lengths from C(4) to C(12); and enhancers in which a double bond is substituted for a single bond in the hydrocarbon chain (3-alkenols) from C(5) to C(9). In addition to the main study, an ancillary set of experiments were to be conducted on the partitioning of a surrogate permeant (estradiol) into the intercellular lipid "phase" under E = 10 isoenhancement conditions to assess the extent to which the permeant partition coefficient may contribute to the permeation enhancement. The following were the principal findings of this research. First, there was very good correlation between the E = 10 isoenhancement aqueous enhancer concentrations and K(octanol/water) for all the studied enhancers. Second, the partitioning of the enhancer from the E = 10 aqueous enhancer solution into the HMS SC intercellular lipid "phase" was found to be relatively independent of the molecular characteristics for all studied enhancers, and the partition coefficients also correlated well with K(octanol/water). These results may have the following meanings: both the microenvironment of the enhancer site of action and the SC intercellular lipid "phase" involved in the enhancer partitioning experiments are well mimicked by liquid n-octanol, and the "intrinsic" potencies (as assessed by the equilibrium enhancer concentration in the microenvironment at the site of action) of the enhancers are relatively independent of the molecular characteristics of the studied enhancers. Finally, the estradiol partitioning experiments suggest the permeant partitioning into the HMS SC intercellular lipid "phase" is enhanced around five- to seven-fold when permeation is enhanced ten-fold for most of the studied enhancers; therefore, the enhancement of the permeant partition coefficient rather than the permeant diffusion coefficient seems to be more important in permeation enhancement of the SC barrier lipoidal pathway.

Mechanistic studies of branched-chain alkanols as skin permeation enhancers.

As part of a long-term effort to understand the structure/function relationship between chemical permeation enhancers and skin permeation enhancement, the present study examined the influence of hydrocarbon chain branching on the effectiveness of skin permeation enhancers of the type that possesses a polar group (e.g., the hydroxyl group) attached to a hydrocarbon chain(s). The effects of x-hexanol, x-heptanol, x-octanol, and x-nonanol (where x is the position of the hydroxyl group ranging from 1 up to 5) on the transport of a probe permeant, corticosterone, across hairless mouse skin (HMS) were investigated. Isoenhancement concentrations are defined as the aqueous concentrations for which different enhancers induce the same extent of permeant transport enhancement, E, across the lipoidal pathway of stratum corneum (SC). The isoenhancement concentrations of 2-alkanol, 3-alkanol, 4-alkanol, and 5-alkanol to induce E = 10 were approximately 1.9-, 2.6-, 3.1-, and 3.9-fold higher, respectively, than those of the 1-alkanols of the same molecular formula. This suggested that the branched-chain alkanols have lower enhancer potency than the 1-alkanols of the same molecular formula; the potency decreases as the hydroxyl group moves from the end of the chain towards the center of the enhancer alkyl chain. To further investigate the mechanism(s) of action of the branched-chain alkanols as skin permeation enhancers, the equilibrium uptake of the enhancers into the hairless mouse skin stratum corneum (HMS SC) from aqueous enhancer solutions of E = 10 was determined. The data from these experiments provided a direct measure of the "intrinsic" potency of the enhancer. In the same experiments, the equilibrium partitioning (distribution) of a surrogate permeant, estradiol (E2beta), into the HMS SC was also determined and compared to the partitioning from PBS (no enhancer present). The uptake amounts (micromole/mg SC) for 1-alkanols into the intercellular lipids of the SC were found to be essentially the same at their isoenhancement concentrations. However, at their isoenhancement concentrations, the uptake amounts of the branched-chain alkanols into the intercellular lipids of HMS SC were higher than those of the 1-alkanols. These results support the view that: (1) the intrinsic potencies of the 1-alkanols are essentially the same and independent of their 1-alkyl chain length at their isoenhancement concentrations, (2) the intrinsic potencies of the branched-chain alkanols are lower than those of the normal alkanols, and (3) branching of the alkyl chain reduces the ability of the enhancer to effect lipid fluidization in the SC lipid lamellae at the target site(s). The enhancement effects of the branched-chain alkanols and the 1-alkanols at their isoenhancement concentrations upon E2beta partitioning into the SC intercellular lipids were found to be approximately the same and in the range of five- to eight-fold enhancement. The constancy of this enhancement for E2beta partitioning suggests that the mechanism of enhancement action for the branched-chain alkanols and the 1-alkanols are the same. Additionally, a good correlation of the intercellular lipid/PBS partition coefficients of both the branched-chain alkanols and the 1-alkanols with the n-octanol/PBS partition coefficients was found. This supports the view that the chemical microenvironment of the polar head group and the alkyl group of the studied enhancers at the site of skin permeation enhancer action in the SC lipid lamellae can be represented by water-saturated n-octanol for both the branched-chain alkanols and the 1-alkanols.

Structure-activity relationship for chemical skin permeation enhancers: probing the chemical microenvironment of the site of action.

Studies were previously conducted in our laboratory on the influence of n-alkanols, 1-alkyl-2-pyrrolidones, N,N-dimethlyalkanamides, and 1,2-alkanediols as skin permeation enhancers on the transport of a model permeant, corticosterone (CS). The experiments were conducted with hairless mouse skin (HMS) in a side-by-side, two-chamber diffusion cell, with enhancer present in an aqueous buffer in both chambers. The purpose of the present study was to extend these studies and investigate in greater detail the hypothesis that a suitable semipolar organic phase may mimic the microenvironment of the site of enhancer action, and that the enhancer partitioning tendency into this organic phase may be used to predict the enhancer potency. CS flux enhancement along the lipoidal pathway of HMS stratum corneum was determined with the 1-alkyl-2-azacycloheptanones, 1-alkyl-2-piperidinones, 1,2-dihydroxypropyl decanoate, 1,2-dihydroxypropyl octanoate, n-alkyl-beta-D-glucopyranosides, 2-(1-alkyl)-2-methyl-1,3-dioxolanes, 1,2,3-nonanetriol, and trans-hydroxyproline-N-decanamide-C-ethylamide as enhancers. Enhancement factors (E values) were calculated from the permeability coefficient and solubility data over a range of E values. Comparisons of the enhancer potencies for all studied homologous series and the carbon number of the n-alkyl group revealed a nearly semilogarithmic linear relationship with a slope of approximately 0.55, which is consistent with the hydrophobic effect. Moreover, comparisons of the enhancer potencies of all the enhancers with the n-hexanol-phosphate buffered saline (PBS), n-octanol-PBS, n-decanol-PBS, and n-hexane-PBS partition coefficients showed very good correlations for the n-alkanol solvents but not for n-hexane. This result supports the interpretation that the enhancer potency is directly related to the ability of the enhancer molecule to translocate to a site of action via its free energy of transfer from the bulk aqueous phase to a semipolar microenvironment in the stratum corneum lipid lamella that is well mimicked by water-saturated n-alkanols.

Evaluation needle length and density of microneedle arrays in the pretreatment of skin for transdermal drug delivery.

Solid silicon microneedle arrays with different needle lengths (ranging from 100 to 1100 microm) and needle densities (ranging from 400 to 11,900 needles/cm(2)) were used to penetrate epidermal membrane of human cadaver skin. After this pretreatment, the electrical resistance of the skin and the flux of acyclovir across the skin were monitored. A linear correlation between the acyclovir flux and the inverse of the skin electric resistance was observed. Microneedle arrays with longer needles (>600 microm) were more effective in creating pathways across skin and enhancing drug flux, and microneedle arrays with lower needle densities (<2000 needles/cm(2)) were more effective in enhancing drug flux if the microneedles with long enough needle length (>600 microm). In addition, the microneedle arrays were used to penetrate hairless rat skin in vivo, and the trans-epidermal water loss (TEWL) of the rat skin was measured before and after the pretreatment. Treating rat skin with microneedle arrays of lower needle density and longer needle length was more effective in increasing TEWL. Integrity of the stratum corneum barrier of the penetrated rat skin as measured by TEWL recovered back to its base line level within 24h after the microneedle pretreatment.