Figure 1.
Characterization of rapamycin-loaded PLGA NPs (rapamycin-NPs) in vitro.
(A). Transmission electron microscopy (TEM) image of rapamycin-NPs. (B). Size distribution of the rapamycin-NPs measured by dynamic light scattering analysis (DLS). (C). In vitro cumulative rapamycin release profiles from rapamycin-NPs (•, red) or free rapamycin (▴, blue), both encapsulated in pluronic gel immersed in PBS buffer. Data are presented as mean as mean ). (C). ing analysis (DLS). (C.
Figure 2.
Accumulation of FITC-loaded NPs in cultured smooth muscle cells and in the arterial wall of the injured rat carotid artery after periadventitial application.
(A). Representative fluorescence microscopic images demonstrate timein the arterial wall of the injured rat carotid arteµg FITC/ml) by cultured rat vascular smooth muscle cells (SMCs) (n = 3). Scale bar represents 10 µm. (B). FITC-NPs were applied around the rat carotid artery immediately after injury (see methods). Representative fluorescence microscopic images of carotid arteries demonstrate the in vivo distribution of FITC-NPs (n = 3) (1 mg FITC-NPs in 300 µl pluronic gel/artery). The first panel of B is a low-magnification longitudinal image of the artery showing perivascular application of NPs. Panels 2–4 are images of cross sections. The last panel shows the auto-fluorescence background of laminas. Scale bar represents 120 µm.
Figure 3.
Prolonged inhibitory effects of rapamycin-loaded NPs on S6K1 phosphorylation in vitro and in vivo.
(A). In vitro experiments. Treatment of SMCs with Rapamycin or rapamycin-NPs (15 µg rapamycin for both) is described in detail in Materials and Methods. Panels a and b show the effect of rapamycin-NPs and rapamycin on p-S6K, respectively. Proteins were extracted from SMCs at the indicated time points, and phosphorylated S6K1 (p-S6K1) and S6K1 were measured by Western blot analysis. Panels c and d show the effect of rapamycin-NPs and rapamycin on SMC proliferation (measured by MTT assay), respectively. Quantified data are presented as mean MC proliferation (measured by MTT (* P<0.05). (B). In vivo experiments. Following balloon angioplasty in rat carotid arteries, rapamycin or rapamycin-NPs (300 µg rapamycin for both) were dispersed in 300 µl pluronic gel and applied periadventitially to injured carotid arteries, as described in Methods. Carotid arteries were retrieved 14 days after surgery. Proteins extracted from carotid arteries were subjected to Western blot analysis for phosphorylated S6K1 (p-S6K1) and S6K1. Quantified data are presented as mean njured carotid arteries, as descri (* P<0.05).
Figure 4.
Sustained inhibitory effects of rapamycin-loaded NPs on intimal hyperplasia in balloon-injured rat carotid arteries.
Balloon injury of rat carotid arteries was performed and rapamycin or rapamycin loaded NPs (300 µg rapamycin for both) dispersed in 300 µl pluronic gel was applied periadventitially immediately after vascular injury, as described in Methods. Solvent (DMSO) and NPs alone dispersed in pluronic gel were used as controls. Carotid arteries were retrieved 14 (A) or 28 days (B) after surgery. Sections were then prepared and H&E stained. Top panel shows representative microscopic images of carotid cross-sections from the indicated treatment groups. Bottom panel shows quantification of lumen size, intimal area, and intimal to media ratio (I/M). Data are presented as mean ± SEM from 5 animals in each group (*P<0.05 compared to DMSO control).
Figure 5.
Sustained inhibitory effect of rapamycin-NPs on cell proliferation in balloon-injured rat carotid arteries.
Rat carotid cross-sections were obtained from the same experiments as in Figure 4. Sections were immunostained for Ki67 as described in Methods. Representative microscopic images of Ki67 staining on arteries retrieved 14 days (A) and 28 days (C) after surgery. Arrows point to Ki67 positive cells. Quantification of Ki67 positive cell number per high power field (HPF) on sections retrieved 14 (B) and 28 days (D) after surgery (magnification is 200X). Each bar represents a mean ±SEM of 5 animals (* P<0.05 compared to DMSO control).
Figure 6.
Periadventitial application of rapamycin-loaded NPs does not affect carotid artery reendothelialization after angioplasty.
Rat carotid cross-sections were obtained from the same experiments as in Figure 4. (A) Representative fluorescence microscopic images of CD-31staining (red, marked by arrows) of arteries retrieved 14 or 28 days after surgery. Blue dots are DAPI-stained nuclei. Dashed lines define internal elastic lamina (IEL). (B) Quantification of reendothelialization (CD-31 positive versus total perimeter) on sections retrieved 14 or 28 days after surgery. Each bar represents a mean ±SEM of 5 animals.