DESCRIPTION (provided by applicant): Port wine stains (PWSs) are congenital and progressive malformations of dermal capillaries. Histopathologically, PWSs are characterized by ectatic capillaries with diameters that can range from about 10 µm to as large as ≈ 600 µm, and usually located in depths of ≈ 300-500 µm below the skin surface. PWS is a disease with potentially devastating psychological and physical complications that greatly impairs the quality of life for the afflicted individuals. Currently, the only viable treatment approach is based on laer irradiation, using visible wavelengths, to thermally destroy the abnormal vasculature. However, may stains are resistive to current laser treatment methods since the visible laser irradiation parameters do not achieve the critical core temperature necessary to irreversibly destroy blood vessels, particularly at the deeper skin locations (>300 µm), necessitating many therapeutic sessions to achieve complete fading, if at all. Furthermore, a large segment of patients with moderate to heavy pigmentation (moderate brown to black skin) cannot benefit from laser therapy due to non-specific heating of the overlying epidermis. Our ultimate objective is to develop a laser-based approach that can be used to treat resistive stains, and all patients with PWS regardless of their skin types. Our proposed approach is based on the use of optical vesicles composed of the FDA-approved near infrared (NIR) chromophore, indocyanine green (ICG), encapsulated by membranes derived from erythrocytes. We refer to these vesicles as near infrared erythrocyte-mimicking transducers (NETs). Once activated by NIR laser irradiation, these vesicles transduce the light energy to heat, leading to thermal destruction of blood vessels. We refer to this approach as laser- erythro-therapy (LET). The advantage of NIR laser irradiation is that it allows for deeper penetration of light into the skin, and reduces the isk of non-specific epidermal heating due to reduced absorption by melanosomes. The potential advantages of NETs as an exogenous chromophore to enhance NIR absorption within the blood vessels are in their potentially long circulation time within the vasculature to extend the therapeutic window of time during which LET can be performed, and their expected biocompatibility. The overall subject of this application is to determine the appropriate formulations of NETs, based on identifying the optimal ICG content, diameter, and relative number concentration, that will result in maximum vascular retention time with appropriate optical properties, as well as optimal NIR irradiation parameters that will result in deep vascular injury without non-specific thermal injury to the epidermis. To do so, our approach will be centered upon physical characterizations of various formulation of NETs, quantification of their optical properties, their biodistributions in healthy mice, mathematical models to predict light an temperature distributions in response to NIR laser irradiation due to these formulations, and finally in-vivo NIR laser irradiation studies involving the rabbit earlobe vasculature as an animal model of PWSs injected with such NETs formulations. We will also evaluate the potential toxicity and immunogenic effects of these formulations in mice. Upon completion of this project, we will have identified the appropriate formulations of NETs, and be in a position to use that information in guiding our human experimental studies in the next grant period following this application. This Multi-PI application will be performed under the leaderships of Dr. Bahman Anvari, Professor of Bioengineering at University of California, Riverside (UCR), and Dr. J. S. Nelson, Medical Director of the Beckman Laser Institute and Medical Clinic (BLIMC), University of California, Irvine (UCI). Dr. Anvari will oversee the entire project, and have overall responsibiliy to coordinate and administer the project. Fabrication and characterization of the NETs, biodistribution, toxicity, and immunogenicity studies will be performed under his supervision. He will also work closely with Dr. Nelson's group on quantification of the optical properties, development of mathematical models and in-vivo animal laser irradiation studies. Other key collaborating personnel are David Lo, MD, PhD (UC Riverside) who will provide the expertise in the design of the immunogenic studies in mice, and evaluating those results; Wangcun Jia, PhD (UC Irvine) who will be involved in quantification of optical properties, development of the mathematical models, and in vivo laser irradiation of the rabbits; and Stephen Griffey, PhD, DVM (UC Davis) who will support us in our toxicity evaluations of the NETs using hematological profiling, serum chemistry, and histopathological evaluations.

PUBLIC HEALTH RELEVANCE: Relevance PWS stain is a congenital and progressively deteriorating skin disease with potentially devastating psychological and physical complications that can greatly impair the quality of life for the afflicted individuals. Currently, the only viabe treatment approach is based on laser irradiation using visible wavelengths to thermally destroy the vascular malformation. However, may stains are resistive to such treatment since visible laser irradiation parameters do not achieve the critical core temperature necessary to irreversibly destroy blood vessels, particularly those seated at the deeper skin locations (>300 µm). Furthermore, a large segment of patients with moderate to heavy pigmentation (moderate brown to black skin) cannot benefit from laser therapy due to non-specific heating of the overlying epidermis This project is aimed at developing a new therapeutic approach based on combination of near infrared laser irradiation and optical vesicles composed of the FDA-approved chromophore, indocyanine green (ICG), encapsulated by membranes derived from erythrocytes. We refer to these vesicles as near infrared erythrocyte-mimicking transducers (NETs), and to our approach as laser-erythro-therapy (LET). Using theoretical and experimental approaches, including animal studies, the overall subject of this application is to determine the appropriate formulations of NETs and laser irradiation parameters that will ultimately result in complete clearance of PWS patients regardless of their skin color blood vessels depth. Upon completion of this project, we will have identified the appropriate formulations of NETs, and be in a position to use that information in guiding our human experimental studies in the next grant period following this application.