Transdermal deferoxamine prevents pressure-induced diabetic ulcers
Dominik Duschera,1, Evgenios Neofytoua,1, Victor W. Wongb, Zeshaan N. Maana, Robert C. Rennerta, Mohammed Inayathullahc, Michael Januszyka, Melanie Rodriguesa, Andrey V. Malkovskiyc, Arnetha J. Whitmorea, Graham G. Walmsleya, Michael G. Galveza, Alexander J. Whittama, Michael Brownleed, Jayakumar Rajadasc,2, and Geoffrey C. Gurtnera,2
aHagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, CA 94305; bDepartment of Plastic Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21201; cBiomaterials and Advanced Drug Delivery Center, Stanford University School of Medicine, Stanford, CA 94305; and dDiabetes Research Center, Albert Einstein College of Medicine, New York, NY 10461
Edited* by Bruce D. Hammock, University of California, Davis, CA, and approved November 26, 2014 (received for review July 16, 2014) There is a high mortality in patients with diabetes and severe to vasculogenesis and wound healing (16). The HIF-1α–HIF-1β pressure ulcers. For example, chronic pressure sores of the heels heterodimer binds to the hypoxia responsive element of oxygen- often lead to limb loss in diabetic patients. A major factor un- sensitive genes, including VEGF (14, 15). In diabetes, HIF-1α derlying this is reduced neovascularization caused by impaired function is compromised by a high glucose-induced and reactive activity of the transcription factor hypoxia inducible factor-1 alpha oxygen species-mediated modification of its coactivator p300, (HIF-1α). In diabetes, HIF-1α function is compromised by a high glu- leading to impaired HIF-1α transactivation (17, 18). cose-induced and reactive oxygen species-mediated modification of Our laboratory has previously demonstrated that deferox- its coactivator p300, leading to impaired HIF-1α transactivation. We examined whether local enhancement of HIF-1α activity would im- amine (DFO), an FDA-approved iron-chelating agent currently prove diabetic wound healing and minimize the severity of diabetic in clinical use for the treatment of hemochromatosis (19), cor- ulcers. To improve HIF-1α activity we designed a transdermal drug rects impaired HIF-1α–mediated transactivation in diabetes by
delivery system (TDDS) containing the FDA-approved small mole- preventing iron-catalyzed reactive oxygen stress. Moreover, DFO BIOCHEMISTRY cule deferoxamine (DFO), an iron chelator that increases HIF-1α reduction of free radical formation decreased cell death (20, 21). transactivation in diabetes by preventing iron-catalyzed reactive Collectively, these effects promote wound healing and decrease oxygen stress. Applying this TDDS to a pressure-induced ulcer tissue necrosis in the setting of diabetes (22, 23). Although sys- model in diabetic mice, we found that transdermal delivery of temic delivery of DFO is not a viable therapeutic option for DFO significantly improved wound healing. Unexpectedly, prophy- diabetic patients owing to potential toxicity and short plasma lactic application of this transdermal delivery system also prevented half-life (24), local transdermal drug delivery systems (TDDS) diabetic ulcer formation. DFO-treated wounds demonstrated in- ENGINEERING creased collagen density, improved neovascularization, and reduc- would be very effective for clinical use. tion of free radical formation, leading to decreased cell death. These findings suggest that transdermal delivery of DFO provides a tar- Significance geted means to both prevent ulcer formation and accelerate dia- betic wound healing with the potential for rapid clinical translation. Diabetes is the leading cause of nontraumatic amputations. There are no effective therapies to prevent diabetic ulcer for- wound healing | diabetes | drug delivery | small molecule | angiogenesis mation and only modestly effective technologies to help with their healing. To enhance diabetic wound healing we designed iabetes mellitus affects over 25 million people in the United a transdermal delivery system containing the FDA-approved DStates (1, 2) and costs nearly $250 billion per year (3). Chronic small molecule deferoxamine, an iron chelator that increases diabetic wounds and decubiti are important long-term sequalae of defective hypoxia inducible factor-1 alpha transactivation in di- both diabetes mellitus types 1 and 2 (4). There is a high mortality in abetes by preventing iron-catalyzed reactive oxygen stress. This diabetic patients who develop decubiti (5–7), and owing to pro- system overcomes the challenge of delivering hydrophilic mol- longed disability and the high rates of recurrence these wounds ecules through the normally impermeable stratum corneum and represent an especially severe complication of diabetes (8). This is both prevents diabetic ulcer formation and improves the healing further underscored by the fact that diabetic nonhealing wounds are of existing diabetic wounds. This represents a prophylactic the leading cause of nontraumatic amputations in the United States pharmacological agent to prevent ulcer formation that is rapidly (3, 9–11). As such, there is a clear need for new approaches to ef- translatable into the clinic and has the potential to ultimately fectively manage and treat diabetic ulcers. transform the care and prevention of diabetic complications. The propensity for wound development in diabetes is associated Author contributions: D.D., E.N., V.W.W., M.I., M.B., J.R., and G.C.G. designed research; with a reduced capacity for ischemia-driven neovascularization (12, D.D., E.N., V.W.W., Z.N.M., R.C.R., M.I., A.V.M., M.G.G., and A. J. Whittam performed 13). Hypoxia inducible factor-1 (HIF-1), which consists of a highly research; J.R. and G.C.G. contributed new reagents/analytic tools; D.D., E.N., V.W.W., regulated α-subunit and a constitutively expressed β-subunit, is a Z.N.M., R.C.R., M.I., M.J., M.R., A.V.M., A. J. Whitmore, G.G.W., M.G.G., A. J. Whittam, J.R., critical transcriptional regulator of the normal cellular response and G.C.G. analyzed data; and D.D., M.B., and G.C.G. wrote the paper. to hypoxia, promoting progenitor cell recruitment, proliferation, Conflict of interest statement: G.C.G., J.R., E.N., and M.G.G. are listed on the following patent assigned to Stanford University: Topical and Transdermal Delivery of HIF-1 Modulators survival, and neovascularization (14, 15). In nondiabetics, hypoxia to Prevent and Treat Chronic Wounds (20100092546). α causes stabilization of HIF-1 protein by preventing the normal *This Direct Submission article had a prearranged editor. α rapid proteasomal degradation of HIF-1 . It does this by inhibiting 1D.D. and E.N. contributed equally to this work. the prolyl hydroxylases (PHDs), which hydroxylate specific prolyl 2 To whom correspondence may be addressed. Email: [email protected] or jayraja@ residues on HIF-1α. Without proline hydroxylation HIF-1α is not stanford.edu. – bound by the von Hippel Lindau E3 ubiquitin ligase complex and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. is able to act as a transcription factor for expression of genes critical 1073/pnas.1413445112/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1413445112 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 Fig. 1. Development of a transdermal drug de- livery system for DFO. DFO aggregates with PVP and surfactants to form reverse micelles (RMs). RMs are dispersed in the polymer ethyl cellulose. After re- lease from the polymer matrix the RMs enter the stratum corneum and disintegrate. PVP dissolves and DFO is delivered to the dermis.
Transdermal delivery of DFO is complicated by its relatively within 24 h (Fig. 2G). To test the importance of the micellar high atomic mass and hydrophilicity (25). This prevents it from delivery of DFO, the TDDS was compared with an otherwise penetrating the lipophilic outermost layer of the skin, the stratum identical formulation containing the established universal solvent corneum, without modification. We therefore developed a DMSO (35) instead of the reverse micelle-forming surfactants. matrix-type TDDS that encapsulates DFO with nonionic surfac- DMSO allowed for solubilization of the hydrophilic DFO in the tants and polymers for delivery enhancement (26, 27). Dispersed hydrophobic chloroform, even in the absence of the surfactant within a release-controlling polymer matrix, the polar DFO mol- molecules. Its use resulted in a homogenous mixture of all ecules are enclosed by reverse micelles, which permit delivery of constituents and prevented any component precipitation. How- DFO through the hydrophobic stratum corneum (28, 29). Apply- ever, despite similar in vitro release profiles (Fig. S1), minimal ing this technology to a murine model of diabetic pressure sores we DFO was delivered into the dermis by the TDDS with the al- were able to prevent ulcer formation and improve diabetic wound tered formulation, confirming the importance of reverse micelle healing through reduction of hyperglycemia-induced oxidative encapsulation for successful transdermal delivery (Fig. 2G). For stress, which impairs HIF-1α transcriptional complex formation. both formulations no DFO could be detected in the receptor buffer of the Franz diffusion cell, consistent with the TDDS’s Results acting as an effective localized delivery system. Development of a TDDS for DFO. To overcome the challenges of To further investigate skin permeation of DFO delivered by transdermal delivery, DFO was formulated into a monolithic the TDDS in vivo (n = 3) we assessed the efficacy of two dif- polymer matrix-type TDDS (Fig. 1). This approach combines ferently dosed TDDSs in uninjured diabetic mice. Using HIF-1α reverse micelle encapsulation of DFO by nonionic surfactants up-regulation as a surrogate for effective DFO delivery we found (29, 30) with dispersion in a degradable slow-release matrix (31), that transdermal DFO treatment resulted in a marginally increased which allows for the targeted delivery of DFO molecules to the dermis (26, 32). Specifically, DFO migrates from the TDDS to the skin following application, as demonstrated by SEM (Fig. 2A). Once through the hydrophobic stratum corneum the reverse micelles can then disintegrate in the more hydrophilic, aqueous environment of the dermis. To confirm the morphology of the DFO-encapsulating reverse micelles and to analyze their structural composition, atomic force microscopy (AFM) and Raman spectroscopy imaging of chemical functionalities were performed (Fig. 2 B–E). As expected, AFM analysis showed several topographically similar large objects with aspheroidalshape(Fig.2B). Moreover, AFM phase imaging, which is sensitive to local sample stiffness, visualized objects in the middle of every spheroid with a stiffness much higher than that of the surrounding shell, representing encapsulated DFO molecules (Fig. 2C). On Raman spectroscopy, doughnut-shaped Raman maps of lipids with the overall shape of the micelle shell were detected (Fig. 2 B and D), whereas the DFO signal correlated with stiff clusters in AFM phase imaging (Fig. 2 C and E). Together, these data indicate successful micellar encapsulation of DFO particles.
DFO Release and Permeation Studies in Vitro and in Vivo. We next Fig. 2. Encapsulation and controlled release of DFO by a TDDS. (A)SEM evaluated the release and permeation abilities of the TDDS images of the TDDS at time 0 (Left) and 48 h post skin application (Right). containing 1% DFO in vitro and in vivo (Fig. 2 F–H). Over 14 h Porous structure remains within the polymer after the drug is released to of incubation in buffer solution under continuous shaking the murine skin (Right). (Scale bars, 100 and 20 μm.) (B) AFM showing the to- cumulative amount of drug released by the TDDS gradually in- pography of formed RM. (C) AFM phase imaging demonstrating DFO particles n = inside the RM. (D) Raman spectroscopy showing the lipid shell of the RM. (E) creased ( 3), highlighting the ability of the TDDS to provide μ sustained delivery of DFO (Fig. 2F). To determine the dermal Raman imaging specific for DFO. (Scale bars, 2 m.) (F) DFO TDDS delivery dem- onstrated a sustained drug release in vitro (n = 3). (G) In vitro penetration profile penetration of DFO delivered by the TDDS, in vitro skin per- n = showing the concentration and location of DFO in full-thickness human skin after meation studies were performed ( 3) using a Franz diffusion 24 h TDDS application (n = 3). (H) Application of different TDDS formulations on cell (33, 34). TDDS application to excised full-thickness human the intact skin of diabetic mice revealed an increase in HIF-1α stabilization in skin demonstrated penetration of DFO into the deep dermis a dose-dependent manner. n = 3. All values represent mean ± SEM; *P < 0.05.
2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1413445112 Duscher et al. Downloaded by guest on October 2, 2021 DFO TDDS Treatment Enhances Neovascularization and Dermal Thickness. To further evaluate the positive effects of DFO TDDS treatment on ulcer healing, histological samples were taken upon complete wound closure. Healed DFO-treated diabetic ulcers exhibited sig- nificantly increased neovascularization compared with the vehicle control group, demonstrated by increased CD31 immonostaining (greater than threefold, P < 0.01, Fig. 4 A and B). Further histo- logical examination of the healed wounds showed that DFO TDDS treatment also significantly improved the dermal thickness of healed diabetic ulcers, visualized as increased picrosirius red staining on polarized light images (greater than threefold, P < 0.01, Fig. 4 C and D). These data indicate that DFO not only accelerates wound closure by increasing neovascularization but also effectively improves the quality of the healed skin.
Localized DFO Treatment Effectively Prevents Diabetic Ulcer Formation. To investigate the prophylactic efficacy of DFO we pretreated the Fig. 3. DFO TDDS improves healing of diabetic ulcers. (A) Full-thickness dorsal skin of diabetic mice for 48 h with a DFO TDDS, followed ulcer wounds of diabetic mice treated with a transdermal DFO TDDS for- by removal of the TDDS and ulcer induction as described above mulation or vehicle control (n = 10). TDDS were replaced every 48 h. (B) (Fig. S3C). Macroscopic monitoring of ulcer formation showed Wound-healing kinetics (wound area as a function of time). Wound closure that skin pretreatment resulted in prevention of ulcer formation occurred significantly faster at day 27 in the DFO-treated group versus day and skin necrosis compared with untreated controls (Fig. 5 A and 39 in the vehicle-treated controls (n = 10). (C) VEGF protein levels in ulcers of B, P < 0.01). Histologic analysis confirmed loss of epithelial in- diabetic mice after transdermal DFO treatment for 1 and 2 d, respectively tegrity, destruction of dermal architecture, and a profound in- (n = 3). (D) Evaluation of VEGF protein expression in skin directly underneath the TDDS, adjacent to it, and 5 mm distant (n = 3). *P < 0.01. flammatory response in controls compared with the minimal tissue destruction observed in DFO TDDS-treated skin (Fig. 5C).
HIF-1α transcriptional activation at 0.5% and a significant increase DFO TDDS Attenuates Tissue Necrosis by Decreasing Apoptosis and BIOCHEMISTRY of HIF-1α at 1% DFO (P < 0.05) (Fig. 2H). These data support the Reactive Oxygen Species Stress. Previous evidence suggests that ap- efficacy of the TDDS as a local delivery method for DFO. TDDSs optosis contributes significantly to cell death following ischemia/ with 1% DFO were used for all further in vivo experiments. reperfusion injury (38, 39). To investigate whether DFO treatment attenuates these apoptotic effects we performed analysis of protein DFO Transdermal Treatment Enhances Wound Healing in Diabetic levels of the apoptotic markers cleaved caspase 3 and Bax in DFO Mice. We adapted an established pressure-induced ulcer model pretreated and control wounds. DFO-pretreated mice showed a sig- db db A–C P < (36) for use in diabetic mice ( / leptin receptor-deficient). nificant reduction of both apoptotic markers. (Fig. 6 , 0.05). ENGINEERING Pressure was applied intermittently by placing a ceramic magnet In ischemic tissues, DFO is known to reduce levels of reactive on both sides of a fold of dorsal skin (Fig. S2 A and B), with 6-h oxygen species (ROS), which play a major role in diabetic ulcer ischemia (magnets on)/reperfusion (magnets off) cycles resulting pathogenesis and persistence (40, 41). Therefore, we evaluated in the most consistent ulcer size and healing kinetics (Fig. S2C). the influence of transdermal DFO treatment on superoxide levels With this protocol, skin ulcers with a thick eschar became ap- using DHE immunofluorescent staining (42). We found that parent after 7 d, and the wounds completely healed by day 35 transdermal delivery of DFO resulted in a dramatic decrease of (Fig. S2D). No deaths, infections, or other complications oc- ROS accumulation, consistent with the observed reduction of curred, and in subsequent experiments all diabetic ulcers were apoptosis, skin necrosis, and ulcer formation (Fig. 6D). induced with 6-h ischemia/reperfusion intervals. To examine the efficacy of transdermal DFO application in diabetic wounds we applied either DFO TDDS or vehicle con- trols onto pressure-induced ulcers on the dorsum of diabetic mice. Transdermal treatment was begun 24 h after the last is- chemia/reperfusion cycle and the TDDS was changed every 48 h until complete ulcer healing (Fig. S3A). TDDS delivery of DFO resulted in significantly accelerated healing (Fig. 3 A and B). Complete resurfacing of ulcers occurred by 27 d in DFO-treated mice versus 39 d in untreated mice (P < 0.01, Fig. 3B).
Transdermal DFO Delivery Increases VEGF Expression. One of the main characteristics of the ischemic environment of diabetic wounds is impaired VEGF production, which directly com- promises neovascularization necessary for proper wound healing (18, 37). Therefore, we evaluated whether sustained DFO de- livery increased VEGF expression in diabetic ulcers. Following application of DFO TDDS to fully developed diabetic ulcers Fig. 4. Localized DFO treatment enhances neovascularization and dermal (Fig. S3B) we assessed VEGF protein levels after 24 and 48 h thickness. (A) Upon complete healing, immunohistochemistry was performed for and observed significantly increased VEGF expression at both the capillary endothelial cell marker CD31 (red). Increased vascularity was seen in C P < transdermal DFO-treated diabetic mice. Blue indicates DAPI staining (Scale bars, time points (Fig. 3 , 0.01). Furthermore, consistent with its 10 μm.) (B) Quantification of CD31-positive pixels per high-power field (HPF) (n = efficacy as a local drug delivery system, VEGF up-regulation was 10). (C) Dermal thickness of completely healed wounds was assessed by polar- limited to the treated area, with adjacent and distant skin being ized light microscopy after picrosirius red staining. (Scale bars, 10 μm.) (D) unaffected (Fig. 3D, P < 0.01). Quantification of picrosirius red-positive pixels per HPF (n = 10). *P < 0.01.
Duscher et al. PNAS Early Edition | 3of6 Downloaded by guest on October 2, 2021 application frequency and thereby increase patient safety and comfort as well as drug efficacy, formulations with sustained drug delivery are ideal. Moreover, this approach could easily be adopted because both diabetic foot ulcers and pressure sores occur in very stereotypic locations and are in general 1–10 cm2 in size. Current clinical care for these wounds includes placing of adhesive sheets (i.e., duoderm) to “cushion” these areas (48), so the application of a therapeutic patch would not be a significant departure from current practice. Oxidative stress plays a pivotal role in the development of diabetes complications. The metabolic abnormalities of diabetes cause mito- chondrial superoxide overproduction, resulting in defective angio- genesis in response to ischemia (49). The underlying mechanism of diabetes-induced superoxide-mediated impairments in neovascu- larization is a dysfunction of HIF-1α (17, 18). A molecular option to therapeutically modulate HIF-1α activity is the iron chelator DFO (50–52). DFO has a crucial advantage over drugs, which purely up- regulate HIF-1α levels, such as the PHD inhibitor dimethylox- alylglycine, in that it also has a direct antioxidant effect and is capable Fig. 5. Transdermal DFO treatment prevents ulcer formation in diabetic mice. of reducing the oxidative stress associated with ischemia (53). In (A) Representative photographs of skin after ulcer induction in diabetic mice keeping with this mechanism, DFO has been found to play a pro- pretreated with either DFO or control TDDS. No severe ulcer formation in the tective role during hypoxic preconditioning in brain (51) and heart DFO-treated group. (B) Quantification of control and DFO-treated necrotic area tissue (54) as well as in cutaneous ischemic preconditioning (23). (n = 10). (C) Representative histological H&E-stained tissue sections showing ulcer Consistent with its predicted therapeutic potential we have pre- formation in the vehicle control group (n = 10). *P < 0.01. (Scale bars, 10 μm.) viously demonstrated the efficacy of topical administration of DFO in healing diabetic wounds (18). However, in order for DFO to be used for ulcer prevention it needs to penetrate the unwounded In summary, these findings support the feasibility of using a stratum corneum, a difficult process owing to the innate properties of transdermal DFO delivery system to address impaired diabetic the skin. The stratum corneum is composed of nonliving corneocytes wound healing. We demonstrate the ability to modulate established and a mixture of lipids organized in bilayers (55). The drug transport biologic pathways, which are impaired in diabetic ulcer healing, and across the stratum corneum barrier is limited by the structural and effectively augment tissue repair and restoration. Further, by pro- solubility requirements for solution and diffusion within the lipid phylactically preloading skin with DFO we demonstrate the ability bilayers (55). Transdermal drug delivery can be classified into two to prevent pressure ulcer formation in a diabetic wound model. categories: passive delivery and active, physically enhanced delivery Discussion (56). Passive delivery approaches include ointments, creams, gels, and transdermal patches. The drugs suitable for passive delivery are In this paper we describe the development of a highly effective usually hydrophobic and have a molecular weight <500 Da, allowing TDDS for the treatment and prevention of diabetic wounds. them to pass through the hydrophobic stratum corneum (57). Un- Specifically, transdermal DFO delivery was found to effectively fortunately, DFO has a molecular weight of 560 Da and consists of up-regulate VEGF secretion and accelerate diabetic wound three hydrophilic bidentate oxygen-containing groups, which reduces healing. Moreover, DFO-treated mice exhibited significantly its lipid solubility (58). Collectively, these physical properties prevent increased angiogenesis and dermal thickness as well as reduced it from penetrating intact stratum corneum. apoptosis and ROS formation in a pressure-induced diabetic Therefore, to use DFO to prevent and treat diabetic ulcers in “at ulcer model. More interestingly, pretreatment with a DFO risk” patients the development of a novel drug delivery platform was TDDS effectively prevented ulcer formation in diabetic mice. Because DFO is already FDA- approved, this TDDS has the potential for rapid translation into clinical application for the prevention and management of diabetic ulcers. There are currently no available pharmacologic agents for the prevention of wound development and only one available to ac- celerate healing in existing wounds (becaplermin, PDGF-BB) (43). Unfortunately, an increased cancer risk has been reported in patients treated with becaplermin, and it is not widely used for this and other reasons (44). Surprisingly, simple pressure offloading remains one of the mainstays of both treatment and prevention of diabetic decubiti and ulcers, but the compliance with these approaches in the long term remains low (45). Several other tech- nologies such as silicone-coated foam and hydrocolloids have been used in attempts to reduce the risk of ulcer formation, but none have demonstrated significant efficacy (46). There is thus an emi- nent need for effective pharmacological approaches to address the tremendous health-care burden of chronic diabetic wounds. The therapy of a chronic disease such as a diabetic nonhealing wound requires repeated treatment administration. In the case of Fig. 6. DFO TDDS attenuates tissue necrosis by decreasing apoptosis and ROS stress. (A) Western blot analysis of Cleaved Caspase-3 (Cl Casp.3) and Bax a substance with a short biological half-life such as DFO, the drug proteins. DFO-pretreated mice show a significant reduction of both apo- would have to be administered within short intervals up to several ptotic markers. (B and C) Quantification of Western blot (n = 3). (D) DHE times daily. Frequent wound dressing changes have been shown to immunofluorescent stain for oxidative stress reveals decreased ROS accu- result in increased pain, irritation, and infection risk (47). To reduce mulation (red) in DFO-treated wounds (n = 3). *P < 0.05.
4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1413445112 Duscher et al. Downloaded by guest on October 2, 2021 necessary. Recent innovative approaches for transdermal drug de- molecules (59). To facilitate dermal penetration of the DFO/PVP complexes, livery include both chemical and physical enhancement (55). More reverse micelle-forming nonionic surfactants polysorbate 80 (Tween 80) and aggressive chemical enhancers such as sulphoxides or alcohols im- sorbitan monolaurete 20 (Span 20) (26) were added to the formulation. Fi- prove the delivery efficiency for hydrophilic molecules but are nally, ethyl cellulose was added to form a slow-releasing matrix (27). For the preparation of the drug-release layer we dissolved the two polymers ethyl known to cause skin irritation and erythema (59). Active physical cellulose (3.5% by weight) and PVP (0.5% by weight) with 1% DFO (by enhancers include iontophoresis (60), sonophoresis (61), electro- weight) in chloroform (27) and added the nonionic surfactants Tween 80 poration (62), or more invasive approaches such as microneedles and Span 20 (1% each, by weight) for reverse micelle formation (26, 59). Di- (63), thermal ablation (64), or skin abrasion (65), but owing to n-butylphthalate was used as a plasticizer (30% weight-in-weight of poly- device complexity and high cost they are still experimental (57). A mers) (27). The solution was stirred vigorously until a fine suspension was less invasive, passive method of augmenting stratum corneum per- achieved. This solution was then poured onto a sterile glass Petri dish and meation involves the use of nonionic surfactants (59). Tween 80 and dried at room temperature. The uniform dispersion was cast onto a 2% Span 20 are widely regarded as safe (59), and chemical enhanced (wt/vol) polyvinyl alcohol backing membrane, dried at 40 °C for 6 h, and cut matrix-type transdermal patches are a very cost-effective way to with a 16-mm circular biopsy punch in equal-sized discs. Finally, the finished deliver molecules across the skin barrier (57), making them an at- transdermal delivery system was attached to a contact adhesive (Tegaderm; 3M). For comparison, an alternative TDDS was formulated using the per- tractive approach with high clinical translatability. meation enhancer DMSO instead of nonionic surfactants, and a control Nanoscale vehicles have undergone rapid development in recent formulation containing only vehicle was prepared by making a suspension years and include liposomes, micelles, and reverse micelles (66). of the polymers and surfactants without the addition of DFO. Reverse micelles consist of a hydrophilic core containing the hy- drophilic drug and a hydrophobic outer shell, which combined with AFM and Raman Spectroscopy Imaging. Both Raman and AFM were performed their small particle size makes them an ideal vehicle for penetrating using NTEGRA Spectra combined AFM-Raman system (NT-MDT) (70). AFM im- the hydrophobic stratum corneum (67, 68). The penetration of the aging was performed in tapping mode with commercial high-durability rounded skin barrier is dependent on multiple factors, including particle po- cantilevers (k = 5.4 N/m, R ∼40 nm) at 0.7 Hz. This provided surface topography larity and size. Nanosized particles are more likely to enter more and phase-contrast images to discern stiffness of different areas within the deeply into the skin than larger ones. Zheng et al. (69) have shown micelle particles. Raman confocal scanning was performed in backscattering that gold cores coated with highly oriented, covalently immobilized, geometry with a long-working Mitutoyo objective (100, 0.7 N.A.). The illumination light was 473 nm, and the power was kept at ∼2 mW to lower the possibility spherical nucleic acid nanoparticle conjugates of siRNA completely of sample damage. Raman maps were produced with a step size of 0.5 mm penetrate keratinocytes in vitro, as well as mouse skin and human and 1-s exposure. Gratings (600 g/mm) were used for optimal signal and epidermis only hours after application. If nanoparticle or micro- spectral resolution. The peaks at ∼1,625 cm−1 (integrated spectral intensities BIOCHEMISTRY − particle movement were based strictly on molecular weight, then this 1,575–1,675 cm 1) were attributed to DFO molecules, whereas the CH bands − − would not be possible. Presumably the particles in that report move at 2,800–3,050 cm 1, less the DFO CH peak at 2,927–2,952 cm 1, were at- through skin via unclear mechanisms not entirely restrained by tributed to the lipid molecules (70). Stokes–Einstein principles, as would be the case in the present study. One particular feature of our approach is the specific target- In Vitro Drug Release. DFO TDDS was placed into 10 mL PBS (pH 7.4) main- ing of well-studied molecular pathways in diabetic pathophysi- tained at a temperature of 37 °C and shaken continuously for 14 h (n = 3). The ology to not only treat but also prevent ulcer formation. Our data concentration of DFO was measured by LC-MS (Shimadzu; AB SCIEX) every suggest that preconditioning potential areas of ischemia/reper- hour as previously described (71) (See SI Materials and Methods for details). ENGINEERING fusion can prevent ulcer formation in susceptible tissues. These In Vitro Skin Permeation. For in vitro skin permeation studies a vertical Franz findings have immense clinical importance, because the pre- diffusion cell model was used (n = 3) as previously described (34) (see SI vention of ulcer formation via a simple, topical TDDS would Materials and Methods for details). significantly reduce patient morbidity and health-care costs as- sociated with chronic diabetic wounds. Pressure Ulcer Model and TDDS Application. Twelve-week-old male C57BL/6 + + db/db mice (BKS. CG-M / Lepr
Materials and Methods Statistical Analysis. Statistical analysis was performed using either ANOVA ± See SI Materials and Methods for further information. or an unpaired Student t test (MATLAB). Values are presented as means SEM. P values < 0.05 were considered statistically significant. Design of the TDDS. A monolithic matrix-type transdermal drug delivery system containing DFO dispersed within a biodegradable polymer was ACKNOWLEDGMENTS. The authors thank Prof. Jürgen Stampfl (Technical University Vienna) for insightful discussion of the manuscript and Revanth designed. DFO mesylate salt powder was purchased from Sigma-Aldrich. All Kosaraju for proofreading. Funding for wound healing research in our lab- reagents used were analytic grade. Owing to its hydrophilicity and tendency oratory has been provided by the National Institutes of Health (R01- to crystallize, DFO is especially well suited for delivery complexed with the DK074095, R01-AG025016, and R03-DK094521), the Harrington Discovery polymer polyvinylpyrrolidone (PVP). PVP is known to stabilize drugs in Institute, the Hagey Family Endowed Fund in Stem Cell Research and Re- an amorphous form (27, 33) and to promote permeation of hydrophilic generative Medicine, and The Oak Foundation.
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