Track: Formulation and Delivery - Chemical - Drug Delivery - Nanoparticles
Category: Poster Abstract
Rational Engineering of Polymeric Nanocarriers for Enhancing Selective Delivery to Vascular Endothelium
Purpose: Vascular endothelium is central to the regulatory of numerous physiological functions, such as vascular permeability, blood fluidity, and angiogenesis.1,2 Damages to vascular endothelium have been reported to cause many acute and chronic pathological conditions, including inflammation, thrombosis, and tumorigenesis.1,2 As such, vascular endothelium represents a prime target for therapeutic interventions. Although the importance of endothelium as a therapeutic target has been recognized, currently, there is still a lack of theranostic agents capable of selectively localizing at vascular endothelium. Consequently, enhancement of targeted therapeutic delivery to vascular endothelium has become an important goal in the pursuit of vascular therapeutics. Among the many types of delivery vehicles, polymeric nanocarriers stand out as promising candidates for transporting cargoes to vasculatures due their tunable physicochemical properties and excellent biocompatibility.3 Unfortunately, many polymeric nanocarriers suffer from poor delivery efficacy and endothelial localization, which may be largely attributed to their inability to overcome various biological phenomena occurring in the circulatory system (circulatory barriers), notably non-specific protein adsorption, inefficient endothelial internalization, considerable phagocytic uptake, and rapid blood clearance.4 Therefore, we hypothesized that polymeric nanocarriers with negligible non-specific protein adsorption, specific affinity to endothelium, phagocyte evading capability, and prolonged circulation lifetime could realize more effective delivery and selective localization at vascular endothelium. To this end, we sought to evaluate the interactions between polymeric nanocarriers and biological entities responsible for circulatory barriers to gain a deeper understanding of the interdependent effects of multiple physicochemical properties of nanocarriers in influencing their biological fate.5 This would then be used to inform the rational engineering of polymeric nanocarriers with improved delivery efficacy and selective uptake by vascular endothelium. Methods: We first synthesized three different 60-nm nanoparticles comprising biocompatible polymeric nanocarriers (polystyrene (PS), poly(lactic-co-glycolic acid) (PLGA), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-mPEG)) encapsulating a hydrophobic organic fluorogen with aggregation-induced emission characteristic (AIEgen) (Fig. 1a). AIEgen was used as a model cargo to visualize and track the biological fate of the nanocarriers. We also synthesized PEGylated nanoparticles (DSPE-mPEG encapsulating AIEgen) with three surface functionalities (COOH, OCH3, and NH2) and two sizes (20 and 60 nm). We then evaluated the nanoparticle morphology, size distribution, zeta-potential, optical absorbance, and fluorescence using atomic force microscopy (AFM), electron microscopy, dynamic light scattering (DLS), ultraviolet-visible (UV-Vis) absorbance, and fluorescence spectroscopies, respectively. The nanocarrier lipophilicity was estimated using ChemBioDraw Ultra software. Next, we probed the interdependent effect of nanocarrier lipophilicity, zeta-potential, and size on the adsorption of plasma proteins (albumin, globulin, and fibrinogen) on nanoparticles using DLS, AFM, SDS-PAGE, UV-vis absorbance and circular dichroism (CD) spectroscopies, and molecular docking simulation. We subsequently assessed the in vitro biocompatibility of the PEGylated nanoparticles using MTT assay and uptake by HUVECs endothelial cells, HeLa epithelial cells, and RAW 264.7 macrophages using confocal microscopy and flow cytometry. Finally, the in vivo biodistribution and circulation lifetime of the PEGylated nanoparticles were evaluated in a zebrafish model. Results: Nanoparticles prepared with PS, PLGA, and DPSE-PEG nanocarriers had a size of about 60 nm (Fig. 1b), negative zeta-potential, and a core-shell structure (Fig. 1c). PS nanocarrier was the most lipophilic while DSPE-PEG nanocarrier was the most hydrophilic (Fig. 1d). Nanoparticles prepared with PS nanocarrier exhibited the most protein adsorption, while those encapsulated within DSPE-PEG had negligible protein adsorption (Fig. 1e-g), suggesting the crucial role of nanocarrier lipophilicity in influencing protein recruitment. After verifying the inertness of DSPE-PEG nanocarrier towards plasma proteins, PEGylated nanoparticles with three surface functionalities, two sizes (Fig. 1h), negative zeta-potential (Fig. 1i), and similar lipophilicity (Fig. 1j) were prepared. Negligible protein adsorption was exhibited by the 20-nm nanoparticles, while for the 60-nm nanoparticles, the least negatively-charged nanoparticles recruited the most proteins (Fig. 1k-m). This indicates that for a given lipophilicity, the nanoparticle size and zeta-potential are instrumental in determining protein adsorption. Further characterization of the nanoparticle-protein associations using AFM, SDS-PAGE, UV-vis absorbance, CD spectroscopies, and molecular docking simulation confirmed our observations. Interestingly, irrespective of nanoparticle size, the more negatively-charged PEGylated nanoparticles were more preferentially endocytosed by HUVECs (Fig. 2a). All PEGylated nanoparticles were biocompatible (Fig. 2b). They were minimally internalized by epithelial cells and macrophages, suggesting the specific affinity of the negatively-charged PEGylated nanoparticles towards endothelial cells and their ability to escape phagocytic uptake. The more negatively-charged nanoparticles also displayed a longer in vivo circulation lifetime and could localize more specifically at vascular endothelium (Fig. 2c-e). Conclusion: In conclusion, sub-100-nm polymeric nanoparticles capable of overcoming circulatory barriers to realize improved delivery and selective localization at vascular endothelium could be rationally engineered through precise optimization of nanocarrier lipophilicity and zeta-potential (Fig. 3). Our proposed two-step design framework could be readily translated into the formulation of advanced polymeric nanocarriers for in vivo theranostics of vascular and other relevant diseases.