Chapter 17 Controlling the In Vivo Activity of Wnt Liposomes

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Controlling the In Vivo Activity of Wnt Liposomes L. Zhao,1 S. M. Rooker,1 N. Morrell,1 P. Leucht,1 D. Simanovskii,2 and J. A. Helms1 Contents 1. Introduction 2. Materials, Methods, and Results 2.1. Manufacture and functional characterization of the phase transition of chromophore-modified liposomes 2.2. Using vesosomes as delivery vehicles for liposomes 2.3. DiI liposome penetration and Wnt3a liposome in vivo activity experiment 3. Concluding Remarks References

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Abstract Liposomes offer a method of delivering small molecules, nucleic acids, and proteins to sites within the body. Typically, bioactive materials are encapsulated within the liposomal aqueous core and liposomal phase transition is elicited by pH or temperature changes. We developed a new class of liposomes for the in vivo delivery of lipidmodified proteins. First, we show that the inclusion of a chromophore into the liposomal or vesosomal membrane renders these lipid vesicles extremely sensitive to very small (mJ) changes in energy. Next, we demonstrate that the lipid-modified Wnt protein is not encapsulated within a liposome but rather is tethered to the exoliposomal surface in an active configuration. When applied to intact skin, chromophore-modified liposomes do not penetrate past the corneal layer of the epidermis, but remain localized to the site of application. Injury to the epidermis allows rapid penetration of liposomes into the dermis, which suggests that mild forms of dermabrasion will greatly enhance transdermal delivery of liposome-packaged molecules. Finally, we demonstrate that topical application of Wnt3a liposomes rapidly stimulates proliferation of cells in the corneal layer, resulting in a thicker, more fibrillous epidermis. 1

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Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, California, USA Hansen Experimental Physics Laboratory (HEPL), Stanford University School of Medicine, Stanford, California, USA

Methods in Enzymology, Volume 465 ISSN 0076-6879, DOI: 10.1016/S0076-6879(09)65017-5

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2009 Elsevier Inc. All rights reserved.

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1. Introduction The physical and chemical properties of liposomes have made them popular vehicles for the delivery of drugs, nucleic acids, and small molecules. Liposomes appear to be particularly well suited for the transdermal delivery of agents to treat dermatologic conditions (de Leeuw et al., 2009). The penetration of most molecules through the epidermis is limited by the corneal layer, which consists of apoptotic keratinocytes organized into keratin-rich, hydrophobic envelopes, and lipid bilayers with hydrophilic regions in between. As a consequence of its hydrophobic nature, the corneal layer allows the preferential penetration of lipid-soluble, hydrophobic molecules over hydrophilic molecules (Bos and Meinardi, 2000). Strongly hydrophobic compounds are hampered from passing through the corneal layer by the hydrophilic regions; consequently, the optimal penetration of molecules through the epidermis requires a careful balance between hydrophobicity and hydrophilicity (reviewed by de Leeuw et al., 2009). Liposomal penetration through the skin is enhanced by dermabrasion, which removes the epidermis but leaves the underlying dermis intact (Friedman and Lippitz, 2009). Even if a helpful drug can pass through the abraded epidermis, the next challenge is to package the reagent in such a way as to avoid degradation in the protease-rich injury site (Toriseva and Kahari, 2009). The packaging method must also not impede the biological activity of the intended drug. Here, we investigated the transdermal delivery of a lipid-modified protein to reduce scarring and stimulate hair regeneration. We describe the engineering of liposomes containing Wnt3a, then show that treatment with Wnt liposomes stimulates activity in the dermis. Wnt proteins act as long-range, concentration-dependent morphogens, so-called because of their abilities to specify patterns of cellular responses based on their tissue distribution (Cayuso and Marti, 2005; Logan and Nusse, 2004). Wnt signals are involved in stem cell self-renewal and proliferation (Nusse et al., 2008) and in adult tissue regeneration (Leucht et al., 2008), and thus may be particularly relevant in dermatologic regenerative medicine. For example, Wnt signals are essential for hair (DasGupta and Fuchs, 1999; Narhi et al., 2008) and skin ( Jia et al., 2008; Nguyen et al., 2006; Ohtola et al., 2008) development, Wnt signaling is activated after skin wounding (Morrell et al., 2008; Widelitz, 2008), and there is a growing literature demonstrating that Wnt signals are necessary for skin wound repair (Fathke et al., 2006; Ito et al., 2007; Ohtola et al., 2008). Lipid modification of the Wnt protein is required for in vivo activity (Mann and Beachy, 2004; Nusse, 2003; Willert et al., 2003). The fatty acid tail appears to be required for cellular secretion and transport (Kadowaki et al., 1996; van den Heuvel et al., 1993), and may allow the Wnt/Wg protein to remain associated with a lipid bilayer during intercellular

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signaling (Zhai et al., 2004). Furthermore, this membrane association may facilitate localization of the Wnt protein at its target receptors, allowing the protein to reach a threshold level required for biological activity (Miura et al., 2006). For instance, there is evidence that the Drosophila Wnt homolog, Wingless, is transported across long distances in a membranebound form (Greco et al., 2001). This theoretical transport mechanism hints at an appealing opportunity: if the endogenous method of lipidated Wnt transport involves association with some sort of lipid raft (Eaton, 2006; Nusse, 2003; Panakova et al., 2005), then perhaps liposomal packaging can be used for the delivery of purified Wnt protein in vivo. Liposomes can form single (unilamellar) or multiple (multilamellar) lipid bilayers that surround an aqueous core. The amphiphathic nature of liposomes allows a hydrophilic molecule to be encapsulated in the aqueous core (de Leeuw et al., 2009). For example, considerable time and energy has been invested in adjusting the physical properties of liposomes, such as lipid bilayer composition, to improve both drug encapsulation and drug release in response to mild hyperthermic conditions (Kakinuma et al., 1996). In an effort to prolong their circulating half-life, ‘‘stealth’’ liposomes have been developed (Cattel et al., 2003). The addition of polyethylene glycol to the exoliposomal surface appears to allow these stealth liposomes to avoid detection by the reticuloendothelial system (Immordino et al., 2006). We postulated that the amphiphathic arrangement of liposomes could be exploited for the in vivo delivery of hydrophobic molecules. We reasoned that the lipid-modified nature of Wnt (Willert et al., 2003) and Hedgehog (Pepinsky et al., 1998) proteins would effectively tether the proteins to the exoliposomal surface of the liposome. This postulated mechanism coupled with dermabrasion, would facilitate the delivery of these molecules to the dermis. While other studies have tracked the distribution of fluorescent molecules after dermabrasion (Watt and Collins, 2008), our study is the first to combine dermabrasion with the liposomal delivery of a lipid-modified protein. Perhaps the single greatest challenge in using liposomes as drug delivery vehicles is controlling when and where the liposomal ‘‘payload’’ is released. In an effort to restrict the site of biological activity, thermosensitive liposomes and localized heating have been employed. Heating causes the lipid bilayer to become more permeable, and agents contained in the aqueous core are released at the site. Heating can be achieved using a waterbath (Kono, 2001) or lasers (Ebrahim et al., 2005; Kim et al., 2007). In previous work we demonstrated that laser sources can trigger phase transition of liposomes in vivo, without causing harm to cells in the heated area (Kim et al., 2007). In this chapter we describe a method to enhance this phase transition while simultaneously reducing the risk of unintended thermal damage to adjacent cells and tissues. This is accomplished by the addition of chromophore-modified lipids into the liposomal membrane. Due to their lipophilic nature, 1,10 -dioctadecyl-3,3,30 ,30 -tetramethylindocarbocyanine

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perchlorate ( DiI) and 1,10 -dioctadecyl-3,3,30 ,30 -tetramethyl-indotricarbocyanine iodide ( DiR) can be directly incorporated into lipid vesicles (Gullapalli et al., 2008). We reasoned that the addition of chromophores to a liposome would effectively create a larger margin of thermal safety in vivo: in effect, the thermal energy delivered to tissues would be preferentially absorbed by the chromophore rather than by the cells. This preferential absorption could then lead to liposome phase transition without inadvertent thermal injury to surrounding tissues. We describe the results of these experiments and our conclusions, which illustrate a unique and precedent approach to the in vivo delivery of Wnt and other lipid-modified proteins and their selective activation via external energy sources.

2. Materials, Methods, and Results Liposomes are prepared using 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in chloroform or 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) in chloroform, both from Avanti Polar Lipids, Inc. (Alabaster, AL). D-Luciferin is obtained from Sigma-Aldrich (St. Louis, MO). VybrantÒ DiI cell-labeling solution (1 mM ) and ‘‘DiR’’ DiIC18 (7) are purchased from Invitrogen Molecular Probes (Eugene, OR). Indocyanine green (ICG) (100 mg) is obtained from TCI America (Portland, OR). Purified mouse Wnt3a protein in 1% CHAPS þ 0.5 M NaCl in 1 PBS (20 mg/ml) is obtained from R. Nusse (Willert et al., 2003) and is added to liposomes as described previously (Morrell et al., 2008). The lipid-modified Wnt preferentially associates with the lipid fraction during preparation (Morrell et al., 2008). An LSL cell-based reporter assay is then used to determine the biological activity of Wnt3a liposomes. LSL cells (ATCC Global Bioresource Center, Manassas, VA) are stably transfected with a construct containing three copies of the TCF-binding site driving luciferase expression in response to exogenous Wnt signals (Ishitani et al., 1999). LSL cells also constitutively express b-galactosidase. Therefore, relative luciferase activity can be normalized against cell number. Both luciferase and b-galactosidase are measured using the Dual-LightÒ Combined Reporter Gene Assay System (Applied Biosystems, Foster City, CA) using a luminometer (Kim et al., 2007).

2.1. Manufacture and functional characterization of the phase transition of chromophore-modified liposomes Thermosensitive liposomes are fabricated in such a way that the phospholipid membrane changes from a gel phase to a liquid-crystalline phase once the critical temperature is reached (Kuo et al., 2000; Needham and

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Dewhirst, 2001). While this method allows liposomes to release their content upon heating, it also has a distinct disadvantage: both the liposome and the surrounding tissue are subjected to the same heating. This issue becomes especially important when the liposome drug target (e.g., tumor) is located deep within the body; in these cases the external temperature must be very high in order to generate hyperthermic conditions at the preferred site of drug release. Since many cellular components, including DNA and vital enzymes, are sensitive to elevated temperatures (Kassahn et al., 2009), even mildly hyperthermic conditions required to activate liposomal drug release can be potentially detrimental to cells and tissues. To avoid these potential complications, we reasoned that the incorporation of chromophores into the liposomal membrane would provide a thermal margin of safety. In effect, the chromophores would act as an ‘‘antennae’’ for thermal energy, selectively absorbing and concentrating thermal energy at the liposomal surface. This selective absorption by the chromophores reduces the amount of heat required to activate liposomal phase transition and spares adjacent cells from thermal damage. Chromophore-modified liposomes are prepared by adding DiI or DiR at 1.5–2% molar amounts to 14 mmol of DMPC (unless otherwise noted). The chromophore and lipid mixture are dried under nitrogen gas in a 10-ml round bottom flask, wrapped in tin foil to preserve the integrity of the chromophore. After drying, 1.0 ml 1 PBS is added to the flask and the mixture is vortexed vigorously until no film remains on the sides of the flask. The mixture is extruded 30 times through a 100-nm polycarbonate membrane positioned in a thermo-barrel extruder (Avanti Polar Lipids, Inc.) to create the chromophore-labeled liposomes. The liposomes are centrifuged at 14,000 rpm for 30 min at 4  C, after which the supernatant is removed and the pellet is resuspended in 0.5 ml of 1 DMEM. These chromophoremodified liposomes are centrifuged are protected from light by storage in foil-covered eppendorf tubes at 4  C. In a separate series of experiments volumetric liposomes are generated, by encapsulating ICG into the liposomal (aqueous) core. Lipid drying is conducted as described above. After drying, 0.284 mmol of ICG (2% of lipid concentration) is dissolved in 1 ml of 1 PBS and added to the lipid-lined flask. The solution is vortexed vigorously until no lipid is observed on the flask, and the lipid-ICG solution mixture is extruded as described above, followed by centrifugation and resuspension in 0.5 ml of 1 DMEM. Volumetric (ICG) liposomes are stored as described above. The phase transition of both chromophore-modified liposomes and volumetric liposomes can be induced through several methods of heating. For these experiments we use a titanium:sapphire (Ti:S; Spectra Physics Tsunami) laser source. A similar laser source has been employed by our group to elicit liposomal phase transition in vivo (Kim et al., 2007).

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After testing a variety of conditions we found that a 1-ns pulse duration, 10 J/cm2 and adjustable light range from a Ti:S laser was sufficient to activate chromophore-modified and volumetric liposome phase transition. We used an acoustic signal, generated when the liposomes rupture (Wu et al., 2008), as a proxy for identifying the point of liposomal phase transition. The acoustic signal intensity, as determined by hydrophone recording, increased in proportion to the energy of the laser (Fig. 17.1A). DiR liposomes exhibited the highest acoustic signal whereas volumetric ICG liposomes produced the lowest acoustic signal when exposed to 350 mJ of energy from a Ti:S laser (Fig. 17.1B). For all three types of chromophore-modified liposomes, we demonstrated that the force of liposomal rupture increased as the energy of the laser increased. ICG and DiR/DiI chromophores are excited at different wavelengths; therefore not all chromophore-modified liposomes responded to a specified laser intensity in the same manner. For example, DiR-modified liposomes showed the largest increase in rupture intensity relative to baseline (Fig. 17.1B) when compared to ICG- and DiI-modified liposomes (Fig. 17.1B). Thus, our data demonstrated that DiR-modified liposomes were more sensitive to laser heating than other forms of chromophoremodified liposomes. These results also raised the possibility that combinations of chromophore-modified liposomes might be useful for sustained and/or sequential drug delivery: by varying the intensity of an external energy source, the selective rupture of one class of chromophore-modified liposomes might be achieved while the integrity of another class of liposomes is maintained.

2.2. Using vesosomes as delivery vehicles for liposomes The inclusion of chromophores into a liposomal membrane is a useful method to enhance phase transition and the release of molecules from the aqueous liposomal core. For hydrophobic molecules that are tethered to the liposomal membrane, however, activity is not dependent on phase transition (Morrell et al., 2008). Rather, molecules positioned on the exoliposomal surface are already accessible to cells in the environment (Morrell et al., 2008). Therefore, liposomes in which the ‘‘payload’’ is already positioned in an active configuration (i.e., on the external surface of the liposome) must be contained within some other vesicle first. In a second step, their activity can be triggered by release from this vesicle. A vesosome is a larger version of a liposome that has room in its aqueous core to harbor liposomes (Kisak et al., 2004). We tested the idea that Wnt liposomes could be packaged into chromophore-modified vesosomes and that these vesosomes could be induced to undergo a phase transition and release the active Wnt liposomes. As a proof-of-principle, we first generated luciferin-containing liposomes (Kim et al., 2007) and then packaged these liposomes into vesosomes.

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Figure 17.1 Liposome membrane phase transition is induced by laser heating. (A) The acoustic signal from the hydrophone measures pressure fluctuations associated with liposomal membrane phase transition during laser heating. The laser pulse is seen at time ¼ 0.00, and the phase transition is measured microseconds later with a sudden increase in amplitude of the acoustic signal caused by liposomal membrane disruption. (B) The amplitude of the hydrophone signal at the time of liposomal membrane phase transition is compared with the energy of the laser for liposomes modified with DiR, DiI, and ICG chromophores. In all groups, the intensity of the acoustic signal increases as the energy of the laser is increased. DiR-modified liposomes show the greatest change in acoustic signal amplitude as laser energy increases and ICG volumetric liposomes show the least change as laser energy increases.

By encapsulating luciferin in the core of the liposome, we could follow the phase transition and stability of the liposome/vesosome combination. Liposomes that contain a 0.3% solution of D-luciferin are generated as described (Kim et al., 2007). After preparation, free luciferin is removed by

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dialysis in 1 PBS overnight. The luciferin liposomes are suspended in 1 DMEM, stored at 4  C, and remain stable for up to 3 weeks (data not shown). Vesosomes are made by drying 15 mmol of DPPC in chloroform with nitrogen gas and then redissolving the lipids in 0.5 ml of HEPES buffered saline solution; ethanol is added to a final concentration of 3 M. The solution is allowed to sit for 2 h and is then transferred to a 15-ml Falcon tube containing 3 ml 1 DMEM. This mixture is centrifuged three times at 3000 rpm for 5 min. Excess ethanol, which is contained in the supernatant, is removed and 185 ml of luciferin liposomes are added, following which, the solution is briefly vortexed. The mixture is then heated to 46  C for 20 min under agitation and centrifuged at 2500 rpm for 5 min. The supernatant is removed and the remaining pellet is resuspended in 0.5 ml 1 DMEM. As before, chromophore-modified liposomes and vesosomes are protected from light. We tested the ability of luciferin liposomes to undergo a selective phase transition in response to external energy (Fig. 17.2). For all of these experiments, an 805-nm laser with a 1-ns exposure is used, generating 10 13 mJ. As expected, luciferin liposomes that lacked a chromophore did not undergo phase transition in response to laser exposure: we found no significant differences in luminescence between luciferin liposomes exposed to the laser and those that were not (p-value ¼ 0.05; Fig. 17.2A). The inclusion of a chromophore in the liposomal membrane, however, rapidly and reliably induced a liposomal phase transition. When exposed to 10 13 mJ, the DiR-modified liposomes showed an increase in luminescence, compared to unheated DiR-modified luciferin liposomes ( p-value ¼ 0.008; Fig. 17.2B). These results demonstrate that the inclusion of a chromophore into a liposomal membrane dramatically enhances the phase transition in response to very small doses of energy. The stability of the vesosomes is tested by encapsulating luciferin-containing liposomes in the vesosomal core. In this configuration, the stability of both the vesosomal and liposomal membranes are tested. We found no significant increases in luminescence of these vesosomes compared to unheated controls ( p-value ¼ 0.04; Fig. 17.2C). These data confirm that our vesosomal preparation provided a stable method for packaging liposomes. In the third series of experiments, the permeability of the vesosomal membrane alone is tested. To do this, DiR-modified luciferin liposomes are packaged into vesosomes whose membranes lacked any chromophores, and as before, the vesosomes are exposed to 10 13 mJ of laser energy. Compared to unheated control, the heated samples showed only a slight increase in luminescence ( p-value ¼ 0.02; Fig. 17.2D). These data show that only chromophore-modified liposomes underwent a phase transition in response to 10 13 mJ; there was, however, a small amount of leakage of the hydrophilic luciferin out of the vesosomal membrane.

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Figure 17.2 The inclusion of a chromophore in the membrane allows targeted activation of liposomal and vesosomal phase transitions. 4T1 cells, which are stably transfected with the firefly luciferase gene (Thorne et al., 2006), were maintained near confluence; various compositions of liposomes and vesosomes were added to the cells, following which the cells and liposome/vesosomes were exposed to an 805-nm laser for 1 ns. Luciferase activity was measured by a luminometer. (A) In the absence of a chromophore, heating of luciferin-containing liposomes did not elicit a significant change in luciferase activity. (B) In the presence of the DiR chromophore, heated luciferin liposomes exhibit significantly greater luminescence than unheated luciferin-containing liposomes. (C) In the absence of a chromophore, heating of vesosomes containing luciferin liposomes produced a modest change in luciferase activity relative to unheated controls. (D) Inclusion of the DiR chromophore in the liposomal surface resulted in moderately higher luminescence compared to unheated controls. (E,F) The inclusion of a DiR chromophore in the vesosomal membrane resulted in higher luminescence compared to unheated controls. Including DiR chromophores in both liposomal and vesosomal membranes resulted in the largest increase in luminescence, compared to unheated controls. Luc lp, luciferin liposomes; luc lp þ DiR, luciferin liposomes with DiR; luc vs, luciferin vesosomes; (luc þ DiR)vs, luciferin liposomes with DiR in vesosomes; luc vs þ DiR, luciferin liposomes in vesosomes with DiR; and (luc þ DiR) vs þ DiR, luciferin liposomes with DiR in vesosomes with DiR. Error bars indicate standard deviation.

In the final set of experiments, the ability of laser heating to trigger the release of liposomes from the vesosomal core is tested. As before, luciferin liposomes are placed into the vesosomal core. In one case (Fig. 17.2E), the

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vesosomal membrane alone contains a chromophore; in the other case, both the liposomal and vesosomal membranes contain chromophores (Fig. 17.2F). In both cases we found that 10 13 mJ was sufficient to induce a phase transition, which was reported by an increase in luminescence after laser heating ( p-value ¼ 0.0001, Fig. 17.2E; p-value ¼ 0.001, Fig. 17.2F). In the latter case, the inclusion of a chromophore into both membranes enhanced the increase in luminescence after exposure to the laser. Our experiments with DiR-modified vesosomes demonstrate that vesosomes can be manufactured in such a way as to encapsulate liposomes. The inclusion of a chromophore and brief exposure to laser heating can selectively trigger the release of these liposomes from their vesosomal compartment. This method of release can be used for both hydrophilic and hydrophobic liposomal molecules, since hydrophilic molecules can be contained in the aqueous core of the vesosome and hydrophobic molecules can be incorporated into the liposomal membrane. We envision such a method could be employed for the simultaneous delivery of a hydrophilic Wnt agonist and the hydrophobic Wnt protein. In the next series of experiments, the extent to which heat treatment affects Wnt liposome activity is tested. Wnt liposomes are fabricated as described previously (Morrell et al., 2008) and then heated for 1 min in a 41  C incubator. This duration and extent of heating produced no significant change in Wnt activity (Fig. 17.3). Thus, the activity of Wnt liposomes does not depend upon liposomes undergoing a phase transition. These data confirm that, rather than being contained in the aqueous core, Wnt proteins are positioned on the external liposomal surface.

2.3. DiI liposome penetration and Wnt3a liposome in vivo activity experiment In the next series of experiments, how the penetration of liposomes into intact and abraded skin is investigated. For these purposes we generated DiI-modified liposomes as described above. The skin injury model consists of a 4-mm diameter injury; the edges of the abraded skin are prevented from retracting by the placement of an O-ring sutured to the intact adjacent epidermis. DiI-modified liposomes are prepared as described above. Between 1 and 2 ml of DiI-modified liposomes are topically administered immediately after wounding, and the treated skin is collected 20 h postsurgery. Shaved, intact skin is treated with 1–2 mL of DiI-modified liposomes served as a control. Hoechst 33342 stain is added to visualize cell nuclei, and the liposomes are viewed under fluorescent light (excitation wavelength 360-340 nm). We discovered that when applied to intact skin, DiI-modified liposomes remained concentrated at the epidermis (Fig. 17.4A). When the tissue was viewed using differential interference contrast (DIC) imaging, the extent of

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Figure 17.3 The activity of Wnt liposomes is not dependent upon phase transition. LSL cells were treated with Wnt liposomes, and then subjected to heating; controls were maintained at 37  C, below the critical temperature for phase transition. Heated and unheated Wnt liposomes exhibited similar levels of activity as measured by luminescence. These data demonstrate that the Wnt protein is maintained in an active configuration that does not require liposomal phase transition, and that elevated temperatures do not decrease the activity of the protein. Error bars indicate standard deviation.

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Figure 17.4 Liposomal penetration into skin can be monitored with the inclusion of a chromophore in the membrane. Intact skin was treated with DiI-modified liposomes, and then visualized under appropriate conditions. (A) Topically applied DiI liposomes (red) do not penetrate through the corneal layer of the epidermis; rather, they accumulate within the epidermis and are maintained at the site of application for at least 20 h. (B) Histology of the skin including the epidermis, dermis and subcutaneous fat shown by DIC microscopy. (C) After injury, DiI liposomes (red) rapidly penetrated into the dermis and the subcutaneous fat layer. e, epidermis; d, dermis; scf, subcutaneous fat.

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penetration could be confirmed as extending no further than the corneal layer (Fig. 17.4B). This finding is in keeping with published reports (Bos and Meinardi, 2000). When the skin was wounded, however, DiI-modified liposomes readily penetrated into the dermis and subcutaneous fat (Fig. 17.4C). The liposomes were still detectable within the wound site 20 h after initial delivery. These data demonstrate that the corneal layer of the epidermis is a barrier to liposome penetration, and that wounding the skin permits liposome penetration as deep as the subcutaneous fat layer, even if only a single application is used. In the last series of experiments, the effects of Wnt liposomes on intact skin are tested. Wnt liposomes are prepared as previously described (Morrell et al., 2008). All liposome preparation materials, including flasks, syringes, and extrusion apparatus are thoroughly washed in concentrated acid detergent, 70% ethanol and deionized water. Briefly, 14 mmol of DMPC are dried in chloroform with nitrogen gas in a 10-ml round bottom flask, and the lipid film is dried overnight in vacuum. PBS is used to dilute stock Wnt3a protein to 1.3 mg/ml. One milliliter of 1.3 mg/ml Wnt (or 1 PBS for PBS liposomes) is added to the lipid-lined flask. The lipid-lined flask is vortexed vigorously until the solution is cloudy, and no lipid remains on the sides of the flask. The cloudy protein-lipid solution is extruded 30 times through a 100-nm polycarbonate membrane in a thermo-barrel extruder (Avanti Polar Lipids, Inc.) held at 30–32  C. After extrusion, the liposomes are centrifuged at 14,000 rpm for 30 min at 4  C to pellet the Wnt3a liposomes from free Wnt3a protein in solution. The supernatant containing free Wnt3a protein is aspirated and the Wnt3a liposome pellet is resuspended in 0.5 ml 1 DMEM. The liposomes are stored at 4  C when not in use. To quantify Wnt liposome activity, a LSL cell-based luciferase assay is conducted as previously described (Morrell et al., 2008). Mouse LSL cells (25,000 cells/well) are plated in 1 DMEM, 10% FBS, 1% penicillin/ streptomycin for overnight incubation at 37  C and 5% CO2. The cells are then treated with varying volumes (5, 10, and 15 ml, each done in triplicate) of purified Wnt3a (3.25 mg/ml) and Wnt3a liposomes. After treatment, the cells are incubated overnight again. PBS liposomes and 1 DMEM treatments are used as baseline controls, and the well volume does not exceed 150 ml. Wnt3a activity is measured by the Dual-LightÒ Combined Reporter Gene Assay System and relative luciferase units are normalized over b-galactosidase activity. Axin2LacZ/þ reporter mice (Jho et al., 2002) are used to visualize Wnt signaling in vivo. In these reporter mice, Wnt signaling leads to the activation of LacZ, which subsequently results in b-galactosidase production. Xgal staining can then be used to visualize this enzyme production. In our experiments, Wnt and PBS liposomes are topically administered to shaved intact skin on the back of Axin2LacZ/þ mice. Tissues are collected after 5 days; the collected tissue is fixed in 0.4% paraformaldehyde

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overnight before being infused with 30% sucrose for 24 h. We then embed the samples in OCT medium and cryosection (12 mm) on Superfrost-plus slides (Fisher Scientific, Pittsburgh, PA). Xgal staining is performed as previously described (Brugmann et al., 2007). We evaluated the baseline level of Wnt signaling in intact skin. Untreated, intact skin from Axin2LacZ/þ mice exhibited Xgal staining around the hair follicles (data not shown). Intact Axin2LacZ/þ skin treated with PBS liposomes showed the same intensity and pattern of Xgal staining around the hair follicles (Fig. 17.5A). Intact Axin2LacZ/þ skin treated with Wnt liposomes showed a robust increase in Xgal staining in the epidermis (Fig. 17.5B). This increase in Wnt signaling was only detectable in the epidermis, coincident with the location of liposome congregation in the epidermis as visualized by DiI liposomes (Fig. 17.4). Together, these data demonstrate that a single application of Wnt liposomes to intact skin dramatically increases Wnt signaling in the epidermis. Liposomes do not penetrate further than the corneal layer, indicating that the extent of Wnt signal amplification can be effectively limited to the outer layers of skin. We evaluated the physiological response to enhanced Wnt signaling in the epidermis. Intact skin treated with PBS liposomes showed a normal organization in the epidermis (Fig. 17.5C). In contrast, the epidermal layer of liposomal Wnt-treated skin was substantially thicker (Fig. 17.5D). This thicker epidermal layer also exhibited greater cell proliferation (Fig. 17.5F) compared to PBS liposome treated skin (Fig. 17.5E). Finally, Wnt liposome treated skin displayed substantially more fibrillous matrix than skin treated with PBS liposomes (Fig. 17.5G and H).

3. Concluding Remarks The therapeutic potential of Wnt proteins for the treatment of dermatologic conditions is enormous. A growing literature implicates Wnt signaling in the development and homeostasis of skin and hair (reviewed by Watt and Collins, 2008), and in the role of Wnt signaling in skin wound healing (Fathke et al., 2006; Ito et al., 2007). Purified Wnt protein, however, does not show sufficient biological activity to induce physiological changes in vivo. The incorporation of Wnt protein into liposomes provides a biomimetic method for exogenous Wnt delivery that results in enhanced Wnt responsiveness in tissues, and a subsequent physiological response in keeping with the role of Wnt signaling in epidermal stem cell self-renewal and proliferation ( Jia et al., 2008; Watt and Collins, 2008). The development of a Wnt liposome delivery system with robust in vivo activity is a critical step toward actualizing Wnts as therapeutic agents.

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Figure 17.5 Topical application of Wnt liposomes to intact epidermis elicits a biological response, stimulates cell proliferation, and enhances birefringent fibril composition in the epidermis. Equal volumes of PBS or Wnt liposomes were topically applied onto the intact skin of Axin2LacZ/þ reporter mice. (A) On postapplication day 5, baseline Xgal staining was detectable around the hair follicles; no obvious Xgal staining was detectable in the epidermis. This pattern of Xgal staining was indistinguishable from untreated control skin. (B) In contrast, treatment with Wnt liposomes elicited robust Xgal staining in the epidermis; Xgal staining in the deeper hair follicles was unchanged from (A). (C) Histological tissue sections reveal the thin, flattened epidermis present in PBS-treated skin. (D) Treatment of intact skin with Wnt liposomes elicits a dramatic thickening of the epidermis. (E) BrdU labeling indicates proliferation of cells around the hair follicle, and minimal proliferation in the corneal layer of the epidermis. (F) In contrast, liposomal Wnt treatment of intact skin resulted in substantially more BrdU-positive cells in the epidermis. Cell proliferation around the hair follicle was unchanged. (G) When visualized under polarized light, picrosirius red staining highlights birefringent fibrils; note the conspicuous lack of picrosirius red staining in the mature, thin epidermis. (H) Liposomal Wnt treatment produced a thicker epidermis, and picrosirius red staining indicated a greater amount of fibrillous matrix within that thicker epidermis (asterisks).

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We also show that the addition of chromophores to the liposomal and vesosomal surfaces offers a novel and highly sensitive method for inducing the phase transition of liposomes. The employment of a laser to induce heating is also an innovative advancement in drug delivery because heating can be produced with precise spatiotemporal control, and with only brief (ns) exposure times. This selective heating method significantly improves the thermal margin of safety and allows targeted liposomal activation without causing undue disruption to cells in the target vicinity. Future experiments will be directed toward optimizing the stability and controlling the activity of Wnt liposomes in vivo.

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