RESUMEN
Topical treatment of vitreoretinal diseases remains a challenge due to slow corneal uptake and systemic clearance. Exosomes are emerging nanocarriers for drug delivery due to biocompatibility and cellular targeting properties. To apply them for retinal targeting via the topical route, exosomes must traverse various ocular barriers including the cornea, lens, vitreous humor (VH), and the retina itself. Here we engineered high-purity milk-derived exosomes by anchoring arginine-rich cationic motifs via PEG2000 lipid insertion on their surface. Modification enabled exosomes to use weak-reversible electrostatic interactions with anionic glycosaminoglycan (GAG) and water content of the tissue to enhance their transport rate and retention. Addition of cationic motifs neutralized the anionic surface charge of exosomes (-24 to -2 mV) without impacting size or morphology. Cationic-motif-modified exosomes exhibited two-fold faster steady state diffusivity through bovine corneas compared to unmodified exosomes. Fluorescence recovery after photobleaching confirmed that cationic-motif-modified exosomes can diffuse through VH without steric hindrance. In healthy VH, cationic-motif-modified exosomes demonstrated stronger binding resulting in three-fold lower average diffusivity that enhanced by six-fold in 50% GAG-depleted VH recapitulating advanced liquefaction. Cationic-motif-modified exosomes penetrated through the full-thickness of porcine retinal explants resulting in ten-fold higher uptake in photoreceptors and three-fold greater transfection with encapsulated eGFP mRNA compared to unmodified exosomes. Cationic-motif-modified exosomes are safe to use as they did not adversely affect the mechanical swelling properties of the cornea or lens nor impact retinal cell viability. Cationic-motif-modified exosomes, therefore, offer themselves as a cell-free nanocarrier platform for gene delivery to retinal photoreceptors potentially via the topical route.
Asunto(s)
Exosomas , ARN Mensajero , Animales , Exosomas/química , Exosomas/metabolismo , Bovinos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Cationes/química , Células Fotorreceptoras de Vertebrados/metabolismoRESUMEN
Multidrug resistance (MDR) observed in tumors significantly hinders the efficacy of chemotherapy. Downregulation of efflux proteins, such as P-glycoprotein (P-gp), using small interfering RNA (siRNA) can be an effective way to minimize the resistance in tumors. In this study, monoclonal antibody 2C5 (mAb 2C5)-PEG7k-DOPE conjugates were post-inserted into the mixed dendrimer micelles containing generation 4 (G4) polyamidoamine (PAMAM)-PEG2k-DOPE and PEG5k-DOPE. The inherent amphiphilic nature of DOPE conjugates causes the copolymers to self-assemble to form a micelle, which can encapsulate hydrophobic chemotherapeutic drugs in its core. The siRNA electrostatically binds to the cationic charges on the G4 PAMAM dendrimer. The tumor-specific mAb 2C5 on the surface of these nano-preparations resulted in improved tumor targeting. This active targeting to tumors can cause increase in the drug and siRNA accumulation at the tumor site, and thereby minimizing the off-target effects. The micelles were shown to have higher cellular association and effectiveness in vitro. The immunomicelle preparation was also tested for cytotoxicity in breast (MDA-MB-231) and ovarian (SKOV-3TR) MDR cancer cell lines.