Personalized Nanotherapy by Specifically Targeting Cell Organelles To Improve Vascular Hypertension

Abstract

Ciliopathies caused by abnormal function of primary cilia include expanding spectrum of kidney, liver, and cardiovascular disorders. There is currently no treatment available for patients with cilia dysfunction. Therefore, we generated and compared two different (metal and polymer) cilia-targeted nanoparticle drug delivery systems (CTNDDS), CT-DAu-NPs and CT-PLGA-NPs, for the first time. These CTNDDS loaded with fenoldopam were further compared to fenoldopam-alone. Live-imaging of single-cell-single-cilium analysis confirmed that CTNDDS specifically targeted to primary cilia. While CTNDDS did not show any advantages over fenoldopam-alone in cultured cells in vitro, CTNDDS delivered fenoldopam more superior than fenoldopam-alone by eliminating the side effect of reflex tachycardia in murine models. Although slow infusion was required for fenoldopam-alone in mice, bolus injection was possible for CTNDDS. Though there were no significant therapeutic differences between CT-DAu-NPs and CT-PLGA-NPs, CT-PLGA-NPs tended to correct ciliopathy parameters closer to normal physiological levels, indicating CT-PLGA-NPs were better cargos than CT-DAu-NPs. Both CTNDDS showed no systemic adverse effect. In summary, our studies provided scientific evidence that existing pharmacological agent could be personalized with advanced nanomaterials to treat ciliopathy by targeting cilia without the need of generating new drugs.

Keywords: Cilia targeting; calcium; drug delivery; hypertension; nanoparticles; nitric oxide.

Figure 1.

Synthesis and characterization of cilia-targeted nanoparticle drug delivery systems (CTNDDS). (a) Fluorescence microscopic imaging of DR-5 localization on primary cilia. Green, red, and blue colors represented acetylated-α-tubulin (ciliary marker), red fluorescence (DR-5), and DAPI (cell nuclei). (b) TEM images. (c) Hydrodynamic size distribution/DLS and (d) zeta-potential of DAu and PLGA NPs before and after different surface functionalizations. (e) SDS-PAGE image showing the incorporation of DR-5 antibody to the DAu and PLGA NPs. The bar graph shows the DR-5 antibody concentrations in the pre- and postconjugation solutions quantified by measuring the absorbance at 280 nm. (f) Fenoldopam-loading and (g) fenoldopam-releasing profiles of CT-DAu-NPs and CT-PLGA-NPs. (h) Photographs showing the synthesized powders of functional CT-DAu-NPs and CT-PLGA-NPs and their dispersion forms in distilled water. n = 3 for all experiments; DR-5 localization was performed in 3 independent experiments from three separate coverslips.*p < 0.05. Statistical analysis was performed using ANOVA followed by a Bonferroni post hoc test.

Figure 2.

In vitro fluorescence imaging of cilia targeted nanoparticles. (a) DIC and fluorescence microscopic imaging of live cells perfused with CT-DAu-NPs (upper panel) and CT-PLGA-NPs (lower panel) for 2 h of time at a constant flow speed. Representative line graphs showing the binding capacity of CT-DAu-NPs (left panel) and CT-PLGA-NPs (right panel) to the cilia and cell membrane. Fluorescence NPs were measured in intensity per area (I/μm²). (b) Representative fluorescence imaging showing the cilia when treated with different treatments, and their length measurements were represented in the bar graph. Green, red, and blue colors represented acetylated-α-tubulin (ciliary marker), red fluorescence (cCT-DAu-NPs, CT-DAu-NPs, cCT-PLGA-NPs, or CT-PLGA-NPs), and DAPI (cell nuclei). n = 3 for all experiments if not represented in dot plot. ****p < 0.0001. Statistical analysis was performed using ANOVA followed by a Bonferroni post hoc test.

Figure 3.

Cellular calcium (Ca²⁺) and nitric oxide (NO) measurements. (a) Fura-2AM ratiometric images showing the changes in the intracellular Ca²⁺ concentrations when cells treated with different treatments under a fluid-shear force of 0.5 dyn/cm². The rainbow color bar indicates the levels of Ca²⁺. (b) DAF-AM ratiometric images showing the changes in the intracellular NO productions when cells treated with different treatments under a fluid-shear force of 0.5 dyn/cm². The green color bar indicates the levels of NO. (c) Single-live cell imaging showing the responses to different treatments. DIC imaging used for tracking a cilium. The induction of flow causes bending of cilium and a subsequent influx of Ca²⁺. The GFP/mCherry ratio (pseudocolored) indicates normalized Ca²⁺ levels. The rainbow color bar indicates Ca²⁺ levels. n = 3 for all experiments.

Figure 4.

Treatment of hypertensive Pkd2 mouse model. (a) Scheme showing timeline for mutation induction and different treatment regimens. TX, tamoxifen. (b) Representative line graphs showing the changes in systolic (SBP) and mean arterial (MAP) blood pressures for 8 weeks. (c) Representative left ventricular pressure-volume (P-V) loops for control, fenoldopam, CT-DAu-NPs, and CT-PLGA-NPs. (d) P-V loops showing the stress response when treated with negative (diltiazem) or positive (epinephrine) chronotropic agents in different treatment groups. (e) Measurements of hearts from control and different treatments of mice were performed using electrocardiograms (ECG). Arrows indicate abnormal spacing. n = 3 for all experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to wild-type vehicle. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 compared to Pkd2 vehicle. Statistical analysis was performed using a second order quadratic polynomial goodness of fit followed with ANOVA using a Tukey’s multiple comparisons test.

Figure 5.

Improvement of biochemistry and heart phenotypes in Pkd2 mice model. (a) Nitrate/nitrite (NOx) and blood urea nitrogen (BUN) concentrations were measured. (b) To assess the heart hypertrophic effect, the thickness of the left ventricle was compared in whole-heart-cross sections using HE staining. Representative microscopic images of HE-stained sections of the left ventricle (LV), showing disparate pathological changes with different treatments. Representative microscopic images of Masson-trichrome-stained sections of LV; myocytes, stained red; collagenous tissue, stained blue. (c) Representative zoomed microscopic images of Masson-trichrome-stained sections of LV showing the amount of fibrosis, which is indicated as blue color. Representative line graphs showing the % of fibrosis in different treatment hearts. n = 3 for all experiments if not represented in dot plot. * p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to wild-type vehicle. #p < 0.05, ##p <0.01, ###p < 0.001, ####p < 0.0001 compared to Pkd2 vehicle. Statistical analysis was performed using ANOVA followed by a Bonferroni post hoc test.

Scheme 1.

Schematic Illustration of Design and Functional Applications of CTNDDS