Anti-ICAM/-Gal NCs are targeted to and remain in tissue:(a) Mice injected with anti-ICAM/125I–Gal NCs were perfused at 30 minutes

Anti-ICAM/-Gal NCs are targeted to and remain in tissue:(a) Mice injected with anti-ICAM/125I–Gal NCs were perfused at 30 minutes. lysosomal transport of anti-ICAM/-Gal NCs in macro- and micro-vascular ECs, and a marked enhancement of Gb3 degradation. Therefore, ICAM-1-targeting strategy may help improve the efficacy of therapeutic enzymes for Fabry disease. Keywords: Vascular endothelium, Fabry disease, ICAM-1 targeting, polymer nanocarriers, -Gal enzyme replacement therapy INTRODUCTION Fabry disease is usually a lysosomal storage disorder (LSD) caused by a genetic deficiency of -galactosidase A (-Gal) [1]. This enzyme hydrolyzes terminal -D-galactosyl residues from neutral glycosphingolipids transported into lysosomes from your blood, plasma membrane and intracellular compartments [1]. As a result of the enzyme deficiency, blood group B substances, galabiosylceramide, and mainly globotriaosylceramide (Gb3) accumulate in body fluids and tissue lysosomes [1]. Gb3 deposits are found in endothelial, perithelial, and smooth-muscle cells in the vasculature, cells of the reticuloendothelial and myocardial tissues, renal epithelial cells, and perineural cells of the autonomic nervous system, causing multi-organ dysfunction and premature death [1]. 1alpha, 24, 25-Trihydroxy VD2 The clinical manifestations of Fabry disease are 1alpha, 24, 25-Trihydroxy VD2 variable, yet life-threatening complications generally arise from progressive cerebrovascular, cardiac and renal impairments caused by the prominent vasculopathy [1]. Vascular lesions lead to myocardial ischemia, hypertension, atherogenesis, stroke, aneurysm, thrombosis, and renal failure [1]. The lung function can also be compromised by airflow obstruction, edema, and pulmonary embolism [1]. These effects of the vasculopathy common of Fabry disease are strongly associated with endothelial dysfunction, and hence, endothelial cells (ECs) are a main target for therapeutic intervention of this malady [1C3]. An available treatment for Fabry disease is usually enzyme replacement therapy (ERT) using recombinant galactosidases that contain mannose-6-phosphate (M6P) [4]. They can bind to M6P receptors on the surface of cells and be transported to lysosomes via clathrin-coated pits [5, 6]. When injected in the blood circulation, these enzymes accumulate in tissues and attenuate Gb3 levels [7C10]. However, despite obvious power of ERT, patients show varying effects and modest response to vasculopathy 1alpha, 24, 25-Trihydroxy VD2 in the cardiovascular and neurological systems [11, 12]. Altered expression or SIR2L4 function of M6P receptors in cells affected by LSDs and formation of immune-complexes that impair enzyme binding to cell receptors may contribute to limit ERT end result [13C16]. Efficacy of ERT for Fabry disease may benefit from strategies enhancing enzyme delivery to organs and vascular ECs via M6P-independent pathways. Targeting of -Gal to intercellular adhesion molecule 1 (ICAM-1) may help accomplish this goal. ICAM-1 is 1alpha, 24, 25-Trihydroxy VD2 usually a transmembrane glycoprotein and an adhesion molecule for leukocytes in inflammation [17]. It is expressed on vascular ECs and other cells in the body, and its expression is usually 1alpha, 24, 25-Trihydroxy VD2 upregulated under pathology, including Fabry disease [18, 19]. ICAM-1 can be targeted by antibodies and affinity peptides for delivery of protein conjugates, contrast and therapeutic agents, and drug delivery vehicles such as liposomes and polymer nanocarriers in cells and animals [20C31]. We have shown that ICAM-1-targeted nanocarriers efficiently enhanced delivery of acid sphingomyelinase (ASM, a lysosomal enzyme deficient in types A-B Niemann-Pick disease) to mouse organs and macrovascular ECs [23, 26, 27]. ASM trafficked to lysosomes by cell adhesion molecule- (CAM)-mediated endocytosis, bypassing clathrin-mediated uptake utilized by current ERTs [27]. Yet, the efficacy of this strategy to enhance the delivery of -Gal or other enzymes and the degree of such enhancement are unpredictable. Different efficacy patterns can arise from potential variations in the enzyme loading capacity of nanocarriers and/or different enzyme pharmacokinetics, which are in part imposed by biochemical properties of the enzyme itself. In addition, delivery of lysosomal enzymes to microvascular ECs (the major endothelial surface in the body, phenotypically and functionally different from macrovascular ECs) [32, 33], has not been tested. Whether ECs with Gb3 storage common of Fabry disease can efficiently internalize and transport nanocarriers to lysosomes also remains to be decided. In this work, we have loaded -Gal on model ICAM-1-targeted nanocarriers (anti-ICAM/-Gal NCs) and used radioisotope tracing, fluorescence and electron microscopy to study this formulation (Calbiochem; San Diego, CA) or coffee bean (Sigma Aldrich; St. Louis, MO) were chosen to distinguish this activity from your endogenous acidic lysosomal counterpart. -Gal from was used in experiments in cell culture. -Gal from coffee bean was used in experiments requiring 125I labeling and in functional activity assays. fluorescein isothiocyanate (FITC)-labeled and non-fluorescent 100 nm diameter polystyrene particles were from Polysciences (Warrington, PA). Cell media and supplements were from Cellgro (Manassas, VA) or Gibco BRL (Grand Island, NY). Na125I and Pierce Iodination Beads were from Perkin Elmer – Analytical Sciences (Wellesley, MA) and Thermo Scientific (Rockford, IL). All other reagents were from Sigma Aldrich (St. Louis, MO). Preparation of anti-ICAM/-Gal nanocarriers and enzyme release Prototype anti-ICAM/-Gal NCs were prepared by adsorbing anti-ICAM or a mix of anti-ICAM and -Gal (95:5 or 50:50 antibody-to-enzyme mass ratio) onto the surface of 100-nm diameter polystyrene particles, as explained [27]. Where indicated, a mix of anti-ICAM and 125I–Gal.

---