Para doenças dermatológicas, incluindo doenças inflamatórias e infecciosas, ex. da leishmaniose e até avaliação estética esse tipo de microscopia não invasiva pode ser útil
In Vivo Imaging of Human and Mouse Skin with a Handheld Dual-Axis Confocal Fluorescence Microscope
Hyejun Ra1,2,9, Wibool Piyawattanametha1,3,4,9, Emilio Gonzalez-Gonzalez1, Michael J Mandella1,2, Gordon S Kino2, Olav Solgaard2, Devin Leake5, Roger L Kaspar1,6, Anthony Oro7 and Christopher H Contag1,8
- 1James H. Clark Center for Biomedical Engineering & Sciences, Department of Pediatrics and the Molecular Imaging Program, Stanford University, Stanford, California, USA
- 2Edward L. Ginzton Laboratory, Department of Electrical Engineering, Stanford University, Stanford, California, USA
- 3National Electronics and Computer Technology Center, Pathumthani, Thailand
- 4Advanced Imaging Research Center, Faculty of Medicine, Chulalongkorn University, Pathumwan, Thailand
- 5Dharmacon Products, Thermo Fisher Scientific, Lafayette, Colorado, USA
- 6TransDerm, Santa Cruz, California, USA
- 7Department of Dermatology, Stanford University School of Medicine, Stanford, California, USA
- 8Departments of Radiology and Microbiology & Immunology, Stanford University, Stanford, California, USA
Correspondence: Hyejun Ra, Department of Pediatrics, Stanford University, Stanford, California 94305, USA. E mail: firstname.lastname@example.org
9These authors contributed equally to this work.
Received 26 August 2010; Revised 27 October 2010; Accepted 1 November 2010; Published online 30 December 2010.
Advancing molecular therapies for the treatment of skin diseases will require the development of new tools that can reveal spatiotemporal changes in the microanatomy of the skin and associate these changes with the presence of the therapeutic agent. For this purpose, we evaluated a handheld dual-axis confocal (DAC) microscope that is capable of in vivofluorescence imaging of skin, using both mouse models and human skin. Individual keratinocytes in the epidermis were observed in three-dimensional image stacks after topical administration of near-infrared (NIR) dyes as contrast agents. This suggested that the DAC microscope may have utility in assessing the clinical effects of a small interfering RNA (siRNA)-based therapeutic (TD101) that targets the causative mutation in pachyonychia congenita (PC) patients. The data indicated that (1) formulated indocyanine green (ICG) readily penetrated hyperkeratotic PC skin and normal callused regions compared with nonaffected areas, and (2) TD101-treated PC skin revealed changes in tissue morphology, consistent with reversion to nonaffected skin compared with vehicle-treated skin. In addition, siRNA was conjugated to NIR dye and shown to penetrate through the stratum corneum barrier when topically applied to mouse skin. These results suggest that in vivo confocal microscopy may provide an informative clinical end point to evaluate the efficacy of experimental molecular therapeutics.
DAC, dual-axis confocal; ICG, indocyanine green; K, keratin protein; MEMS, microelectromechanical systems; NIR, near infrared; PC, pachyonychia congenita; siRNA, small interfering RNA
In vivo imaging has the potential to monitor and assess the effectiveness of skin therapeutics and augment the current state-of-the-art biopsy procedure by providing image guidance and the power of repeated measures. As biopsies are painful and may complicate interpretation of treatment regimens because of their invasiveness and resulting tissue damage, the use of noninvasive microscopy to guide tissue sampling and increase the number of data points can refine the study, and possibly reduce or replace biopsies. Our group has developed a handheld dual-axis confocal (DAC) fluorescence microscope that is capable of detecting individual keratinocytes expressing green fluorescent protein in mouse skin in vivo (Gonzalez-Gonzalez et al., 2010a). This in vivo microscope has also been used to show the effects of small interfering RNA (siRNA) on target gene expression in the skin after intradermal injection (Gonzalez-Gonzalez et al., 2009). This approach provided quantitative data that revealed the temporal inhibition patterns of reporter gene expression in a living transgenic mouse model (Ra et al., 2010).
The ability to monitor noninvasively, at single cell resolution, the effectiveness of siRNA-based or other experimental medicines in the skin would offer an informative measure of therapeutic outcome that would accelerate and refine the development of new treatment strategies for skin disorders, and in the management of these diseases. Recently, the first clinical trial to test the effectiveness of siRNA in skin was completed for the rare skin disorder, pachyonychia congenita (PC), with promising results (Leachman et al., 2010). PC is an uncommon inherited skin disorder caused by dominant-negative mutations in the differentiation-specific keratins, keratin (K) 6a, K6b, K16, or K17, which disrupt intermediate filament function and lead to epithelial cell fragility (McLean and Lane, 1995; Leachman et al., 2005). The symptoms, resulting from defective keratin filament formation in a specific subset of epithelial tissues, include thickened dystrophic nails, painful palmar and plantar hyperkeratosis, leukokeratosis, cysts, and follicular hyperkeratosis (Leachman et al., 2005;Smith et al., 2005). Therapies based on siRNAs have been developed that target the genes and genetic mutations responsible for PC (Hickerson et al., 2008;Leachman et al., 2008; Smith et al., 2008), and one of these, K6a_513a.12 (formulated Good Manufacturing Practice version is called TD101), which targets a single nucleotide mutation (p.Arg171Lys) in the KRT6A gene, was tested in a phase 1b clinical trial (Hickerson et al., 2008; Leachman et al., 2010). The PC patient from the published clinical trial (Leachman et al., 2010) was the subject of the imaging studies presented here.
In this study we demonstrate the capacity of the DAC system to visualize cellular architecture in mouse skin, and then test its ability to evaluate anatomic differences in human skin at different places in the body following topical delivery of near-infrared (NIR) dyes and, finally, to evaluate the ability of NIR dye-conjugated siRNA to penetrate the stratum corneum barrier when applied topically to mouse skin. This is done with the goal of using this technology to noninvasively evaluate siRNA delivery and effectiveness in patient skin, specifically PC skin. In mouse skin, the NIR dye utilized was IRDye 800CW (LI-COR Biosciences, Lincoln, NE)—this agent is being evaluated for clinical use (Marshall et al., 2010). For the human skin studies, a clinically approved dye, indocyanine green (ICG, IC-GREEN; Akorn, Buffalo Grove, IL), was used. Formulated IC-GREEN more readily penetrated callused or PC skin when compared with calf skin, suggesting that the skin barrier properties are compromised in diseased or callused skin and that the dye and DAC microscope may comprise a useful measure of therapeutic outcome.
DAC microscopy can visualize single cells in living mouse skin
The DAC system was used to first demonstrate micro-anatomic imaging in mouse and human skin using a topical dye formulation, and later to show functional imaging of siRNA delivery in murine skin. NIR fluorescent dye (IRDye 800CW) was topically applied to mouse footpad skin to test tissue uptake in vivo. Figure 1shows an en face image at 52 μm depth and a side view of the acquired three-dimensional (3D) tissue volume after topical application of IRDye 800CW in GeneCream formulation. This formulation penetrates through the stratum corneum into lower layers of the epidermis and into the dermis (to a depth of 100 μm), and the IRDye appears to penetrate cells and localize within the cytoplasm, as shown in Figure 1a, where dark nuclei of epidermal keratinocytes are clearly visualized. The use of contrast agents such as IRDye 800CW and ICG enable visualization of tissue microanatomy, and may provide a useful measure of epithelial cell fragility and the resulting reorganization of the ultrastructure of the skin.
Visualization of individual keratinocytes in mouse footpad skin by noninvasive in vivo imaging. Dual-axis confocal (DAC) intravital image stacks were obtained 30–60 minutes after topical application of IRDye 800CW (GeneCream formulation) to mouse footpad skin (full movie file is provided in Supplementary Materials online). (a) En faceimage taken at a depth of 52 μm. Scale bar=50 μm. (b) Side view of the full three-dimensional (3D) volume. The volume dimensions for b are 420 × 155 × 100 μm3.Full figure and legend (112K)
Imaging of normal human skin
In order to evaluate the DAC system for imaging human skin in vivo and to test the ability of the GeneCream formulation to penetrate normal and diseased skin, the NIR handheld DAC microscope and topical application of cream-formulated IC-GREEN was used to noninvasively image the microanatomy of healthy skin. A normal volunteer was imaged at two distinct anatomic sites with differing microanatomy; a site on the calf of the leg and callused plantar region of the foot (Figure 2). Figure 2a and b show en face images of the smooth thin skin of the calf taken at depths of 27 and 55 μm, whereas Figure 2c shows the side view of the full 3D image volume. The dye was observed to penetrate down to a depth of 80 μm in most regions. Beyond the stratum corneum, the cream seemed to consistently penetrate deeper layers of the normal epidermis in an uneven manner (see Figure 2b). Images of a callus region of the foot are shown in Figure 2d–f. En face images at 27 and 55 μm demonstrate that the dye, in comparison with the smooth skin of the calf, is distributed more uniformly throughout the callus area, as shown in Figure 2d and e. Penetration of the dye down to 160 μm depth was observed in some areas of the callus, which was substantially greater than that observed in the calf (Figure 2f).
In vivo imaging demonstrates enhanced topical delivery of near-infrared (NIR) dye to live layers of human plantar callused skin when compared with smooth calf skin in a volunteer. Dual-axis confocal (DAC) image stacks were generated (full movie files are provided in Supplementary Materials online) from volunteer skin after topical application of GeneCream-formulated IC-GREEN NIR imaging dye. (a, b) En face calf skin images at depths of 27 and 55 μm. (c) Side view of the full three-dimensional (3D) volume. (d, e) En faceimages of callused skin taken at depths of 27 and 55 μm. (f) Side view of the full 3D volume. The 3D volume dimensions are 650 × 320 × 150 μm3. Scale bar=50 μm.Full figure and legend (210K)
DAC analysis of PC patient epidermis
Similar imaging procedures were performed with the PC patient who had recently completed a clinical trial with TD101 and vehicle control (Leachman et al., 2010). The siRNA (TD101)-treated foot callus is shown in Figure 3. En face images at 25 and 43 μm depth are presented in Figure 3a and b, whereas a perspective side view of the 3D image stack is shown in Figure 3c. The fluorescence signal was imaged down to a depth of 50 μm, and individual keratinocytes can be observed, probably within the granular layer of the epidermis (Figure 3a and b). Most of the dye remained in the top epidermal layers of the treated skin, and signal was barely detected below a depth of 70 μm. This resembles the nonlesional calf skin of the same patient (data not shown), which was similar to images acquired in the calf skin of the normal volunteer (Figure 2a–c). Images of vehicle-treated plantar callus are also shown in Figure 3. En face images at depths of 25 and 80 μm are presented in Figure 3d and e, whereas a side view of the 3D image stack is shown in Figure 3f. The dye readily penetrated beyond 110 μm in PC symptomatic skin (vehicle treated), sometimes showing a strong fluorescence signal at depths >70 μm (Figure 3e–f). After initial imaging, a region of callus from the vehicle-treated foot was trimmed in order to investigate differences in dye uptake and penetration after a reduction in the amount of callus present. Following debridement, more cells were observed that resembled keratinocytes, such as those shown in dashed circles in Figure 4a, which were located at a depth of 13 μm. The signal was relatively high down to 150 μm in depth, whereas diffuse fluorescence was detected beyond 200 μm in depth. The TD101-treated foot skin was not pared as there was no appreciable callus to trim at the treatment site (Leachman et al., 2010).
In vivo imaging of small interfering RNA (siRNA)- and vehicle-treated pachyonychia congenita (PC) patient skin reveals differences in dye penetration and tissue morphology. Dual-axis confocal (DAC) image stacks were generated (full movie files are provided in Supplementary Materials online) from patient skin 30–60 minutes following topical application of GeneCream-formulated IC-GREEN imaging dye. (a, b) En face images of siRNA-treated callused skin at depths of 25 and 43 μm. (c) Side view of the full three-dimensional (3D) volume. (d, e) En face images of vehicle-treated callus at depths of 25 and 80 μm. (f) Side view of the full 3D volume. The 3D volume dimensions are 650 × 320 × 100 μm3. Scale bar=50 μm.Full figure and legend (194K)
In vivo imaging following trimming of pachyonychia congenita (PC) patient callus and topical application of near-infrared (NIR) dye reveals individual skin cells. Dual-axis confocal (DAC) image stacks were acquired 20–60 minutes after topical application of GeneCream-formulated IC-GREEN imaging agent (complete movie file is provided in Supplementary Materials online). (a) En face image of epidermal cells (inside dashed circles) at a depth of 13 μm. These cells are likely keratinocytes based on morphology and depth. Scale bar=50 μm. (b) Side view of the full three-dimensional (3D) volume of 650 × 320 × 200 μm3.Full figure and legend (157K)
DAC imaging of cutaneous siRNA uptake in vivo
DAC microscopy has the potential to also follow the delivery of therapeutic agents into the skin by using a fluorescent tag on the agent and imaging its distribution. However, these studies cannot be conducted in humans. Therefore, to test the ability of the DAC system to directly visualize delivery of siRNA in skin, we returned to a mouse model. Cream formulated NIR dye-labeled siRNA was applied to murine back skin and the depth of penetration of the therapeutic nucleic acid was assessed. Figure 5 shows in vivo images at 3 hours after topical application. The NIR dye used for conjugation is DyLight 800 (Thermo Fisher Scientific, Rockford, IL), which has similar fluorescence properties to IRDye 800CW. Keratinocytes can be seen in optical sections taken with the DAC microscope, such as inside the dashed circles of Figure 5a at a depth of 20 μm. Unlike the use of the more general contrast agents (ICG and IRDye 800CW) that are intended to stain a substantial amount of tissue to reveal microanatomic changes, imaging of labeled therapeutics should reveal localization of the agent.Figure 5b shows the side view of the 3D tissue volume, where a uniform signal is observed down to 50 μm. This suggested that the labeled siRNA penetrated the stratum corneum and was in association with epidermal keratinocytes.
Intravital imaging demonstrates penetration of small interfering RNA (siRNA) through the murine stratum corneum barrier following topical application. Dual-axis confocal (DAC) image stacks (complete movie file is provided in Supplementary Materials online) were generated 3 hours after topical application of DyLight 800-conjugated siRNA to mouse back skin. (a) En face image at a depth of 20 μm; epidermal cells (likely keratinocytes based on morphology and depth) can be visualized inside the dashed circles. Scale bar=50 μm. (b) Side view of the full three-dimensional (3D) volume with dimensions of 405 × 235 × 70 μm3.Full figure and legend (217K)
The NIR DAC system features a miniaturized handheld microscope that acquires cellular-resolution images in real time, which is enabled by the dual-axis architecture (Wang et al., 2003) and microelectromechanical systems (MEMS) technology (Ra et al., 2007). The NIR wavelength of the laser and superior rejection of scattered light allow for deep imaging in tissue that is relevant not only for normal skin but also for a variety of skin pathologies. In addition, the 3D imaging capability of the microscope provides perspective views of tissue volumes that are otherwise difficult to attain from traditional tissue sections. These characteristics of the DAC system in its miniaturized package make in vivoimaging of cellular and molecular changes possible through the range of preclinical studies to clinical applications, especially in areas that are difficult to reach with larger, static imaging systems.
In this study, we demonstrated that topically applied fluorescent agents to mouse and human skin allow intravital visualization of individual cells and measurement of the penetration depths of contrast agents with the DAC microscope, and this can be used to assess transdermal delivery. IRDye 800CW delivered with the GeneCream formulation exhibits cytoplasmic localization in mouse skin, whereas Food and Drug Administration (FDA)-approved IC-GREEN was used in place of IRDye 800, which has similar fluorescence attributes in the NIR region of the spectrum, for imaging patient skin. We observed that nonaffected skin in a PC patient and noncallused skin in a normal volunteer (e.g., calf skin) were more impervious to dye delivery and therefore are more difficult to access via topical application. The dye distribution was generally nonuniform, and some of the dye may have penetrated through compromised regions of the skin. This pattern continued down to a depth of 80 μm, whereas at greater depths, only a diffuse signal was observed (see Figure 2b and c). In contrast, in vehicle-treated, callused PC skin or calluses of a normal volunteer, the GeneCream formulation readily penetrated the stratum corneum and delivered IC-GREEN to greater depths. This may be because of a “loosening” of the “morter” and/or tight junctions between stratum corneum keratinocytes, resulting in a compromised barrier function, allowing some of the cargo to penetrate further. These results are consistent with previous reports where fluorescently labeled oligonucleotides showed greater penetration through regions of severely impaired stratum corneum formation in ex vivo psoriatic skin, demonstrating nuclear localization in parakeratotic cells, as well as other keratinocytes (White et al., 2002). In contrast, those agents remained confined to the stratum corneum of normal skin. In the PC study shown here, the vehicle-treated, callused skin of the PC patient showed more structural disorganization of the callus, likely because of the expression of filament-disrupting mutant K6a protein (see Hickerson et al., this issue). This can be observed in Figure 3d–f, where the dye appeared to have been transported through fissures within the epidermis and became more widely distributed in the deeper layers of the skin.
Imaging of siRNA-treated PC skin revealed changes suggestive of reversion to healthy nonaffected skin when compared with skin treated with vehicle alone. siRNA-treated PC skin showed comparable dye penetration patterns with healthy skin from a normal volunteer (compare Figure 3a–c with Figure 2a–c). Additionally, DAC imaging of the vehicle-treated foot after trimming of the callus region also revealed live cells of the epidermis (see Figure 4a), although the cells are not as clear as in Figure 1, where IRDye 800CW was utilized. This may be because of lower uptake of the IC-GREEN dye compared with IRDye 800CW, where similar results were observed in mouse skin experiments (data not shown), or it may be explained by the greater quantum efficiency of the IRDye 800CW relative to IC-GREEN. The increased resolution may also be due, at least in part, to differential cellular localization of the dyes; the IRDye 800CW does not appear to enter the nucleus, allowing better resolution of subcellular structures. The nonuniform dye uptake pattern of the trimmed callus region is similar to that of healthy skin of the volunteer, where the majority of signal is concentrated at the upper layers and more diffuse in deeper skin layers. It should also be noted that the in vivo DAC imaging of human skin was a proof-of-principle study with limitations of a small subject group consisting of one control and one patient.
All of the experiments above were focused on in vivo imaging of skin with topically applied NIR dyes to observe and assess differences in tissue structure. As a next step, we have demonstrated that topical cream application of DyLight 800-conjugated siRNA allows for penetration through the murine stratum corneum barrier. The distribution of the dye-conjugated siRNA was mostly uniform throughout the upper layers of the epidermis, which is desirable for clinical translation. Keratinocytes in the upper layers of the epidermis can be observed (see Figure 5a), although little, if any, of the labeled siRNA appeared to enter the cells, as appears to occur with NIR dyes alone (compare with Figure 1). This is likely because of the higher molecular weight and polyanionic nature of siRNA. In recent studies, we have shown that unmodified siRNA is not readily taken up by cells in the absence of pressure and that the so-called “self-delivery” modified siRNAs may facilitate keratinocyte uptake (Gonzalez-Gonzalez et al., 2010a, 2010b). Future experiments using NIR dye-labeled siRNAs that have been modified to enhance cellular uptake, such as the Accell technology from Thermo Scientific/Dharmacon (www.dharmacon.com) (Gonzalez-Gonzalez et al., 2010b) or self-delivery inhibitors being developed by RXi Pharmaceuticals (www.rxipharma.com), may be highly informative. In this context, the DAC system can be used to study various siRNA delivery methodologies using fluorescently tagged siRNAs, which will not only show whether penetration through the stratum corneum barrier is achieved, but also give an idea of whether the siRNA is being taken up by keratinocytes.
In our study of in vivo fluorescence microscopy in the skin with the DAC system, we conclude that 3D cellular imaging with a microscope conducive to preclinical and clinical settings brings value to assessing siRNA uptake and/or differences in tissue structure. This supports in vivo imaging with the DAC microscope as an effective clinical end point in evaluating the efficacy of molecular therapeutics of the skin.
Materials and Methods
In vivo DAC microscopy system
The specifications of the handheld DAC microscope system for in vivo imaging have been previously reported (Ra et al., 2008; Piyawattanametha et al., 2009). The microscope operates at the NIR wavelength of 785 nm, which allows for noninvasive imaging in skin down to a depth of 200 μm. The dual-axis optical configuration (Wang et al., 2003) and MEMS technology (Ra et al., 2007) allow miniaturization of a confocal microscope into a 10-mm-diameter handheld unit that is amenable for intravital imaging as well as clinical imaging in skin. The transverse and axial resolutions are 5 and 7 μm, respectively. The MEMS device continuously scans for real-time en face imaging at 5 frames per second, whereas a piezoelectric actuator translates the MEMS scanner in the depth direction to acquire image stacks. All image stacks start at 10 μm below the skin surface. Full 3D volumes are reconstructed using Amira (Visage Imaging, San Diego, CA) software. The demonstration of the DAC system in animal studies has shown stability and reproducibility in in vivo fluorescence imaging (Ra et al., 2010).
Preparation of GeneCream topical formulations
For the IRDye 800CW formulation (550 μl), 0.5 mg of IRDye 800CW N-hydroxysuccinimide Ester Infrared Dye (LI-COR Biosciences) was dissolved in 30 μl of DMSO, and 10 μl of this solution was mixed with 500 ml of a lipid/alcohol-based cream (“GeneCream”) (Takanashi et al., 2009) to give a final concentration of 0.3 mg ml–1. For the ICG topical formulation, clinical grade IC-GREEN (Akorn) was dissolved in 10 mm Tris (pH 7.5) at 10 mg ml–1 and formulated to a final concentration of 2 mg ml–1 in GeneCream. CBL3 siRNA (targets click beetle luciferase (Gonzalez-Gonzalez et al., 2009)) was synthesized with a 5′N6 modification, a primary amine for the N-hydroxysuccinimide ester reaction, for conjugating the DyLight 800 N-hydroxysuccinimide Ester Infrared Dye (Thermo Fisher Scientific). Following conjugation, excess dye was removed using an anion exchange column, and molecular weight was verified by mass spectrometry. The resulting conjugated compound was used to image siRNA treatment with the DAC microscope.
Cream application and imaging of mice
Before cream application, the skin was cleaned with alcohol (70% isopropanol) swabs (BD, Franklin Lakes, NJ) and allowed to dry for 5 minutes. Mice were anesthetized using 2% isofluorane, and approximately 50 μl of cream was topically applied to the mouse footpad or back skin that was closely shaved with a razor blade (Personna, Hauppauge, NY) 2 hours before cream application. Excess cream was removed from the treated area 30 minutes after application with alcohol swabs and the mice were imaged with the DAC microscope under the same anesthesia conditions. Gel (Vidal Sassoon hair gel, Beverly Hills, CA) was applied to the region of skin as an index-matching optical coupling agent for imaging with the DAC microscope. All animal work was carried out under strict adherence to institutional guidelines for animal care of both the National Institutes of Health and Stanford University.
Cream application and imaging of human skin
The patient and volunteer treatment and imaging were performed with Institutional Review Board approval (Stanford IRB Protocol no. 14823). The study was conducted according to the Declaration of Helsinki Principles, and participants gave their written informed consent. Approximately 200 μl of the IC-GREEN cream formulation was applied to callused and noncallused skin regions (previously cleaned with alcohol swabs) of a normal volunteer or PC lesions (nonaffected, siRNA-treated, or vehicle-treated regions) of a patient who had completed siRNA therapy (Leachman et al., 2010). The patient imaging occurred 48 days after the last siRNA treatment, at which time there was a clear difference in callus thickness between the siRNA-treated and vehicle-treated sites. As a control, the cream was also applied to clinically nonaffected calf skin of the PC patient. Plantar callus of the normal volunteer was imaged as a model for the callus in PC patients, whereas noncallused calf skin of the volunteer was imaged as a model for normal healthy skin. Excess cream was removed with cotton pads 15–30 minutes after topical application. Next, gel (Vidal Sassoon hair gel) was applied to the skin as an optical coupling agent and imaging was performed with the DAC microscope. To facilitate imaging of live skin layers, callused regions were groomed by 3 to 4 mm in depth by the patient using a scalpel, stopping just before penetrating the underlying, innervated, vascularized dermis. Formulated IC-GREEN was subsequently applied and the skin was imaged as described above. Multiple sites of the skin were imaged with the DAC microscope in each area. Representative images are presented in the figures.
Conflict of interest
RLK has intellectual property in the area of using siRNAs to treat skin disorders and topical siRNA delivery and is an employee of TransDerm. MJM and GSK are founders of and consultants to Optical Biopsy Technologies (OBTI) with financial interests in OBTI. DL is an employee of Dharmacon Products, Thermo Fisher Scientific.
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