title: Rapamycin-loaded nanoparticles for inhibition of neointimal hyperplasia in experimental vein grafts.
abstract: BACKGROUND
  Nanoparticles (NPs) possess several advantages as a carrier system for intracellular delivery of therapeutic agents. Rapamycin is an immunosuppressive agent which also exhibits marked antiproliferative properties. We investigated whether rapamycin-loaded NPs can reduce neointima formation of vein graft disease in a rat model.
  
  
  METHODS
  Poly(lactic-co-glycolic acid) (PLGA) NPs-containing rapamycin was prepared using an oil/water solvent evaporation technique. The size and morphology of the NP were determined by dynamic light scattering methodology and electron microscopy. In vitro cytotoxicity of blank, rapamycin-loaded PLGA NPs was studied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Excised rat jugular vein was treated ex vivo with blank NPs, or rapamycin-loaded NPs, and then interposed back into the carotid artery position using a cuff technique. Grafts were harvested for 21 days and subjected to morphometric analysis as well as immunohistochemical analysis and Western blotting.
  
  
  RESULTS
  Rapamycin was efficiently loaded in PLGA NPs with an encapsulation efficiency of 87.6%. The average diameter of NPs was 180.3 nm. The NPs-containing rapamycin at 1 ng/mL significantly inhibited vascular smooth muscular cells proliferation. Measurement of rapamycin levels in vein grafts showed that the concentration of rapamycin in vein grafts at 3 weeks after grafting was 0.9 ± 0.1 μg/g. In grafted veins without treatment, intima-media thickness was 300.4 ± 181.5 μm at 21 days after grafting, whereas veins treated with rapamycin-loaded NPs showed a reduction of intimal-media thickness of 150.2 ± 62.5 μm (p = 0.001). Cell proliferation was measured by proliferating cell nuclear antigen immunohistochemistry staining. As expected, proliferating cell nuclear antigen index declined from 83.4% ± 7.4% to 66.2% ± 4.5% in vein grafts after 3 weeks (p = 0.002). Platelet endothelial cell adhesion molecule (PECAM-1/CD31) staining was used to measure luminal endothelial coverage in grafts and indicated a high level of endothelialization at 21 days after grafting, with no significant effect of blank or rapamycin-loaded NPs group. Western blot analysis showed that treatment with rapamycin-loaded PLGA NPs markedly attenuated phosphorylation and activation of S6 kinase 1 phosphorylation and inactivation of 4E (eIF4E)-binding protein 1, both in vascular smooth muscular cells and vein grafts at 7 and 21 days after grafting.
  
  
  CONCLUSIONS
  We conclude that sustained-release rapamycin from rapamycin-loaded NPs inhibits vein graft thickening without affecting the endothelial cells in rat carotid vein-to-artery interposition grafts; thus, this may be a promising therapy for the treatment of vein graft disease.
authors.affiliation: Division of Vascular Surgery, Department of General Surgery, the First Affiliated Hospital of Nanjing Medical University, Nanjng, Jiangsu Province, China.
authors.forename: Junjie
authors.initials: J
authors.lastname: Zou
authors.forename: Xiwei
authors.initials: X
authors.lastname: Zhang
authors.forename: Hongyu
authors.initials: H
authors.lastname: Yang
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhu
authors.forename: Hao
authors.initials: H
authors.lastname: Ma
authors.forename: Shui
authors.initials: S
authors.lastname: Wang
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002317:Cardiovascular Agents / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics; D002339:Carotid Arteries / Q000601:surgery; D002352:Carrier Proteins / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D003692:Delayed-Action Preparations; D004337:Drug Carriers; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006085:Graft Survival / Q000187:drug effects*; D006965:Hyperplasia; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins; D007601:Jugular Veins / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology / Q000637:transplantation; D019344:Lactic Acid / Q000737:chemistry; D008027:Light; D008297:Male; D046529:Microscopy, Electron, Transmission; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D053758:Nanoparticles; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D011100:Polyglycolic Acid / Q000737:chemistry; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D051381:Rats; D017207:Rats, Sprague-Dawley; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D012542:Scattering, Radiation; D020123:Sirolimus / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics; D012995:Solubility; D017539:Tunica Intima / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology / Q000637:transplantation; D058017:Vascular Grafting / Q000009:adverse effects*
chemical_list: D002317:Cardiovascular Agents; D002352:Carrier Proteins; D003692:Delayed-Action Preparations; D004337:Drug Carriers; C499526:Eif4ebp1 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D019893:Ribosomal Protein S6 Kinases; C526901:Rps6kb1 protein, rat; D020123:Sirolimus