Endothelial-Specific Targeting of RhoA Signaling via CD31 Antibody-Conjugated Nanoparticles

Abstract

Existing Vascular Endothelial Growth Factor (VEGF)-oriented anti-angiogenic approaches are known for their high potency. However, significant side effects associated with their use drive the need for novel anti-angiogenic strategies. The small GTPase RhoA is an established regulator of actin cytoskeletal dynamics. Previous studies have highlighted the impact of endothelial RhoA pathway on angiogenesis. Rho-associate kinase (ROCK), a direct RhoA effector, is potently inhibited by Fasudil, a clinically-relevant ROCK inhibitor. Here, we aimed to target the RhoA signaling in endothelial cells by generating Fasudil-encapsulated CD31targeting liposomes as a potential antiangiogenic therapy. The liposomes presented desirable characteristics, preferential binding to CD31-expressing HEK293T cells and to endothelial cells, inhibited stress fiber formation and cytoskeletal-related morphometric parameters and inhibited in vitro angiogenic functions. Overall, this work shows that the nanodelivery-mediated endothelial targeting of RhoA signaling can offer a promising strategy for angiogenesis inhibition in vascular-related diseases. Significance statementSystemic administration of anti-angiogenic therapeutics induces side effects unrelated to the treatment tissues. We, among others, have shown the impact of the RhoA signaling in the endothelial cells and their angiogenic functions. Here, to minimize potential toxicity, we generated CD31-targeting liposomes with encapsulated Fasudil, a clinically-relevant Rho kinase inhibitor and successfully targeted endothelial cells. In this proof-of-principle study, the efficient Fasudil delivery, its impact on the endothelial signaling, morphometric alterations and angiogenic functions verify the benefits of site-targeted anti-angiogenic therapy.

Characterization of a Nonselective Opioid Receptor Functional Antagonist: Implications for Development as a Novel Opioid Dependence Medication

“…The MTT assay was utilized to assess the cytotoxicity of the test compound following the previously established procedure. ,, In brief, bEnd-3 cells (passage 25–28) (ATCC, Manassas, VA) or Caco-2 cells (passage 21–25) were seeded into 96-well plates (8 × 10 3 cells per well) in 100 μL of complete medium. The complete medium included a mixture of Dulbecco’s Modified Eagle Medium (DMEM) (Fisher Scientific, Waltham, MA, USA), 10% Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA) in the case of bEnd-3 cells and 20% FBS in the case of Caco-2 cells, 1% penicillin–streptomycin (Fisher Scientific, Waltham, MA, USA), and 1% Non-Essential Amino Acids (NEAA) (Fisher Scientific, Waltham, MA, USA).…”

“…The MTT assay was utilized to assess the cytotoxicity of the test compound following the previously established procedure. 6,35,36 In brief, bEnd- Waltham, MA, USA) in the case of bEnd-3 cells and 20% FBS in the case of Caco-2 cells, 1% penicillin−streptomycin (Fisher Scientific, Waltham, MA, USA), and 1% Non-Essential Amino Acids (NEAA) (Fisher Scientific, Waltham, MA, USA). The cells were incubated at 37 °C in a 5% CO 2 humidified incubator for 60 h. Then, the medium was removed, and the wells were washed with 150 μL of Phosphate-Buffered Saline (PBS) (Fisher Scientific, Waltham, MA, USA).…”

Construction Strategy of Functionalized Liposomes and Multidimensional Application

“…Additionally, the specific liposomes exhibited enhanced permeability and longer circulation duration compared to non‐targeted liposomes, thus facilitating effective administration of therapeutic agents to the intended site. [ 124 ] Zhou et al. [ 125 ] used α8 integrin antibody‐modified liposomes to deliver dexamethasone and captopril to achieve targeted anti‐inflammatory/anti‐fibrosis therapy for glomerulonephritis.…”

Mechanistic Aspects of Biphenyl Urea-Based Analogues in Triple-Negative Breast Cancer Cell Lines

“…The experiments were performed following the previously reported procedures. , Briefly, each cell line was seeded at a density of 8 × 10 3 cells per well in a 96-well flat-bottom cell culture plate (cat no. 08-772-2C, Falcon, Fisher Scientific), using the respective complete medium.…”