Intravenous Delivery of Lung-Targeted Nanofibers for Pulmonary Hypertension in Mice

Abstract

Pulmonary hypertension is a highly morbid disease with no cure. Available treatments are limited by systemic adverse effects due to non-specific biodistribution. Self-assembled peptide amphiphile (PA) nanofibers are biocompatible nanomaterials that can be modified to recognize specific biological markers to provide targeted drug delivery and reduce off-target toxicity. Here, PA nanofibers that target the angiotensin I-converting enzyme and the receptor for advanced glycation end-products (RAGE) are developed, as both proteins are overexpressed in the lung with pulmonary hypertension. It is demonstrated that intravenous delivery of RAGE-targeted nanofibers containing the targeting epitope LVFFAED (LVFF) significantly accumulated within the lung in a chronic hypoxia-induced pulmonary hypertension mouse model. Using 3D light sheet fluorescence microscopy, it is shown that LVFF nanofiber localization is specific to the diseased pulmonary tissue with immunofluorescence analysis demonstrating colocalization of the targeted nanofiber to RAGE in the hypoxic lung. Furthermore, biodistribution studies show that significantly more LVFF nanofibers localized to the lung compared to major off-target organs. Targeted nanofibers are retained within the pulmonary tissue for 24 h after injection. Collectively, these data demonstrate the potential of a RAGE-targeted nanomaterial as a drug delivery platform to treat pulmonary hypertension.

Keywords: nanofibers; peptide amphiphiles; pulmonary hypertension; targeted drug delivery.

Figure 1.

Structure and characterization of ACE- and RAGE-targeted PA nanofibers. Chemical structures of (A) ACE-targeted PAs: GNG PA, RYDF PA, and TPTQ PA, and (B) RAGE-targeted PAs: AMV PA, KGVV PA, and LVFF PA. (C) Representative TEM images of all targeted nanofibers reconstituted in 1 mg/mL in HBSS at varying co-assembly molar ratios. Both targeting epitope and co-assembly molar ratios influenced fiber formation. Scale bar: 500 nm.

Figure 2.

Hypoxia-induced pulmonary hypertension in CBL57/6 mice. (A) Hematoxylin and eosin-stained lungs demonstrating increased vessel wall muscularization (black arrows) in hypoxic vs. normoxic mice. Scale bar: 50 μm. (B) The number of non-muscularized, partially and fully muscularized small (25–75 μm) pulmonary vessels were quantified in normoxic vs. hypoxic mice (n=9–10). *P<0.05, **P<0.01, ***P<0.001; Kruskal-Wallis test. (C) Immunofluorescence staining of SMC α-actin (red) in pulmonary vessels (white arrows). Blue = DAPI nuclear staining. Green = lung autofluorescence. Scale bar: 50 μm. (D) Increased SMC α-actin levels indicate hypermuscularization of pulmonary vasculature in hypoxic mice (n=6). ***P<0.001; Mann Whitney test. Echocardiographic findings in normoxic vs. hypoxic mice comparing (E) pulmonary artery acceleration time (PAT), (F) pulmonary artery velocity time index (VTI), and (G) pulmonary vascular resistance (PVR) demonstrate elevated arterial pressures in hypoxic mice (n=11). *P<0.05; paired Student’s t-test. (H) RVSP waveform tracing demonstrates elevated pressures in a mouse with hypoxia-induced pulmonary hypertension. (I) Quantification of RVSP in normoxic vs. hypoxic mice (n=6–11). **P<0.01; 2-sample Student’s t-test. In B, D-G, and I, data are expressed as mean ± SEM and each dot represents an individual animal.

Figure 3.

Increased ACE and RAGE levels in CBL57/6 mice with chronic hypoxia-induced pulmonary hypertension. (A) Representative images of immunofluorescence staining of ACE (red) or RAGE (red) in normoxic vs. hypoxic lungs. Blue = DAPI nuclear staining. Green = lung autofluorescence. Scale bar: 50 μm. Quantification of lung (B) ACE fluorescence intensity (n=6) and (C) RAGE fluorescence intensity (n=7) for normoxic vs. hypoxic mice. Mean ± SEM. *P<0.01, **P<0.001; Mann Whitney test. Each dot represents an individual result; 16 images were analyzed per animal.

Figure 4.

RAGE-targeted LVFF nanofiber localizes to lung with hypoxia-induced pulmonary hypertension. Normoxic and hypoxic mice were injected with ACE- and RAGE-targeted nanofibers. (A) Representative image of 3D LSFM demonstrating fluorescence localization (red) of nanofibers in normoxic vs. hypoxic mouse lungs. Lung autofluorescence is represented in green. Scale bar: 1500 μm. (B) Comparison of normoxic vs. hypoxic mice injected with non-targeted VVAAEE nanofiber (n=6–7), RYDF nanofiber (n=5–7), TPTQ nanofiber (n=5–6), AMV nanofiber (n=5–7), KGVV nanofiber (n=4–5) and LVFF nanofiber (n=5–6). *P<0.05. (C) Distribution of LVFF nanofiber throughout the lung in normoxic and hypoxic mouse lungs (n=5–6). (D) Male and female mice injected with LVFF nanofiber had similar fluorescence levels in normoxic vs. hypoxic conditions (n=7 males, 4 females). Each dot represents an individual result. In B-D, data were analyzed by Kruskal-Wallis test with Bonferroni correction. Mean ± SEM.

Figure 5.

LVFF nanofibers colocalize to RAGE in hypoxic lung. Immunofluorescence staining of RAGE (purple) in hypoxic lungs from 4 mice injected with the LVFF nanofiber (red). Green is tissue autofluorescence. Blue is DAPI stain (nuclei). Scale bar: 100 μm.

Figure 6.

Less targeted epitope incorporated in LVFF nanofiber co-assembly increases localization. (A) Representative images of 3D LSFM demonstrating nanofiber lung localization (red) after injection of non-targeted VVAAEE nanofibers vs. 25, 50, and 75 mole % LVFF nanofiber co-assembly molar ratios in normoxic vs. hypoxic mice. Green is tissue autofluorescence. Scale bar: 1000 μm. (B) Quantification of nanofiber fluorescence in normoxic vs. hypoxic mice injected with non-targeted VVAAEE nanofibers (n=6–7), 25 mole % LVFF nanofibers (n=9), 50 mole % LVFF nanofibers (n=5–6), and 75 mole % LVFF nanofibers (n=6–9). Amongst hypoxic mice, 25 mole % LVFF nanofibers had greater lung localization. *P<0.05, **P<0.01 compared to 25 mole % LVFF nanofibers in hypoxia. All LVFF co-assembly molar ratios had significantly more fluorescence in the hypoxic lung. #P<0.01, ##P<0.001 compared to respective normoxic group. (C) Quantification of 25 mole % LVFF nanofiber localization between upper, middle, and lower regions of the lung in normoxic vs. hypoxic mice (n=9). Results in B and C were analyzed by Kruskal Wallis test with Bonferroni correction. (D) The number of individual 25 mole % LVFF nanofiber fluorescence objects detected per lung volume in normoxic vs. hypoxic mice (n=9) demonstrating that nanofiber accumulation is evenly distributed rather than due to large clumps of nanofibers clustered together. ##P<0.001; Mann Whitney test. (E) Graph demonstrating relationship between 25 mole % LVFF nanofiber fluorescence intensity and nanofiber fluorescence volume in a hypoxic mouse. Scale bar: 70 μm. (F) Male and female mice injected with 25 mole % LVFF nanofiber had similar fluorescence levels in normoxic vs. hypoxic conditions (n=9 males and females, respectively). Data analyzed with Kruskal Wallis test. In B-D and F, data are expressed as mean ± SEM and each dot represents an individual result; 4 images per animal were analyzed.

Figure 7.

Dosing and targeting duration of 25 mole % LVFF nanofiber. (A) Representative images of 3D LSFM demonstrating localization of 25 mole % LVFF nanofiber (red) in hypoxic lungs at 5 mg/kg, 10 mg/kg, and 20 mg/kg. Green is tissue autofluorescence. Scale bar: 1500 μm. (B) Quantification of 25 mole % LVFF nanofiber fluorescence in hypoxic mice at 5 mg/kg (n=7), 10 mg/kg (n=8), and 20 mg/kg (n=9). (C) Timeline of workflow for targeted nanofiber injection followed by return to normal activity until time of sacrifice at 30 min, 4 hrs, and 24 hrs, respectively. At each time interval, lungs were imaged with (D) LSFM to evaluate localization of 25 mole % LVFF nanofiber (20 mg/kg, red). Green is tissue autofluorescence. Scale bar: 1500 μm. (E) Quantification of fluorescence in hypoxic mice at 30 min (n=9), 4 hrs (n=12), and 24 hrs (n=8) after injection with 25 mole % LVFF nanofiber. In B and E, data expressed as mean ± SEM. *P<0.01; Kruskal Wallis test with Bonferroni correction. Each dot represents an individual result; 4 images were analyzed per animal.

Figure 8.

Off-target organ biodistribution of 25 mole % LVFF nanofiber. (A) Representative images of 3D LSFM demonstrating 25 mole % LVFF nanofiber localization (red) to liver, kidney, and heart at 30 min, 4 hrs, and 24 hrs after injection. Green is tissue autofluorescence. Scale bar: 1000 μm. (B) Quantification of off-target localization at 30 min (n=8–9), 4 hrs (n=12), and 24 hrs (n=7–8) in hypoxic mice. *P<0.05 for 4 hr liver vs. all other treatment groups; Kruskal Wallis test with Bonferroni correction. Each dot represents an individual result; 4 images were analyzed per animal. (C) Dose-response curve of average fluorescence intensity (579 nm emission) for standardized amounts of 25 mole % LVFF nanofiber. Red squares represent individual data points and red solid line represents the line of best fit. (D) Amount of nanofiber fluorescence excreted in the urine at 30 min (n=4), 4 hrs (n=8), and 24 hrs (n=7) post nanofiber injection in hypoxic mice vs. non-injected hypoxic controls (n=6). *P<0.001; one-way ANOVA with Tukey’s test. Each dot represents an individual animal. (E) Representative cross-sectional LSFM image of kidney at 30 min vs. 4 hrs after injection with 25 mole % LVFF nanofiber in hypoxic mice. Nanofiber accumulation migrates from the collecting duct (white arrow) to the renal parenchyma as time progresses with minimal remaining at 24 hrs (shown in panel A). Scale bar: 1000 μm. (F) Representative urine sample of hypoxic mice treated with 25 mole % LVFF nanofiber at 30 min vs. 4 hrs after injection. Gross fluorescence is lost by 4 hrs. (G) Cumulative excretion of nanofiber fluorescence over time (30 min, n=4; 4 hrs, n=8; 24 hrs, n=7) compared to non-injected hypoxic controls (n=6). Excretion was calculated as a percentage of the total fluorescence provided by injection of intravenous 25 mole % LVFF nanofiber. In B, D and E, data expressed as mean ± SEM.