Simvastatin-Loaded Chitosan-Functionalized PLGA Nanoparticles: Characterization and Use in Intimal Hyperplasia Therapy

Figures

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  Figure 1. Mevalonate/cholesterol synthesis pathway. Statins inhibit HMG-CoA reductase, leading to inhibition of cholesterol synthesis and isoprenoid intermediates. The inhibition of isoprenoid intermediates disrupts the activation of G proteins, including Ras and Rho.

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  Figure 2. Characterization of SL-cNPs and E-cNPs. Particle size distribution, hydrodynamic diameter (A), PDI (B), and charge (zeta potential, (C)) were determined for E-cNPs, SL-cNPs, and SL-NPs. Data shown as lots around the mean, using 3 separate lots (analyzed using Student’s T-test, with * p < 0.05 as significant). Transmission electron microscopy was utilized to visualize the E-cNPs, SL-cNPs, and SL-NPs at 5000× magnification; bar represents 800 nm (D).

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  Figure 3. SL-cNPs encapsulated with DiD lipophilic tracer readily associate with ECs and VSMCs. Human ECs (a–c) or VSMCs (d–f) were cultured with either basal media or basal media with 0.2 mg SL-cNPs or with 0.2 mg SL-NPs for 30 min. For flow cytometry (a–f), cells were harvested and dispersed into flow cytometry tubes with basal media. SL-cNP- and SL-NP-positive cells elicited an emission wavelength at 664 nm. ECs and VSMCs treated with SL-cNPs demonstrated increased association to SL-cNPs as compared to control cells and cells treated with SL-NPs. Immunostaining was then preformed to visualize association of SL-cNPs to cells (g–j). Cells were fixed and labeled with DAPI (blue) or Alexa Fluor 488 Phalloidin (green). Red fluorescence signifies DiD-encapsulated SL-cNPs (outlined with red circle).

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  Figure 4. SL-cNPs enhance RhoA and RhoB mRNA content in EC and VSMC cultures. (a) Endothelial cells; (b) vascular smooth muscle cells. Confluent ECs or VSMCs were cultured in basal media without or with E-cNPs, free simvastatin, or low-dose or high-dose SL-cNPs for 30 min and then washed. Relative changes in RhoA and RhoB mRNA content were quantified by RT-qPCR. Data shown are means ± SEM (n = 3) and are expressed as GAPDH-normalized fold-changes. * p < 0.05 compared to control; # p < 0.05 compared to low-dose SL-cNPs; § p < 0.05 compared to free simvastatin; statistical analysis performed using ANOVA.

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  Figure 5. Photomicrograph of representative cuts of intimal hyperplasia after balloon injury of carotid arteries. Rats were sacrificed 14 days after balloon injury, and carotid arteries were harvested, perfused, fixed, sectioned, and stained with hematoxylin and eosin. Utilizing autofluorescence, intimal hyperplasia was measured using intimal/medial area ratios. (a) No statin control; (b) empty-cNPs; (c) oral simvastatin; (d) periadventitial SL-cNPs; (e) intraluminal SL-cNPs; (f) intraluminal SL-cNPs and oral simvastatin. Arrows indicate areas of intimal hyperplasia. White bars represent 200 µm.

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  Figure 6. Intima/intima + media ratios. Oral simvastatin in combination with intraluminal administration of low-dose SL-cNP significantly reduced intimal hyperplasia compared to control rats. Data presented as means ± SD. Control (n = 8), E-cNP (n = 7); oral simvastatin (n = 9); periadventitial SL-cNPs (n = 7),; low-dose SL-cNP (n = 7); high-dose SL-cNPs (n = 8); oral + SL-cNP (n = 7). Analysis was performed using one-way ANOVA; *, p < 0.05.