OBJECTIVETo evaluate the feasibility and stability of chemically conjugating IgM on collagen films.

METHODSIgM was labeled with 125I using the chloramine-T method. Six collagen films were randomly divided into two groups. In chemical coupling group 125I-labeled IgM was chemically coupled with the films through N-succinmiclyl-3- (2-pyridyl-dithio) propionate reaction. In control group 125I-labeled IgM was absorbed onto collagen films. The amount of IgM on the collagen films and the amount of IgM remained on the films after extensive rinsing with phosphate buffered saline were monitored by counting the radioactivity of 125I.

RESULTSThe amount of antibodies loaded onto collagen films in the chemical coupling group was 15 times higher than that on the control films, showing significant statistical difference (P < 0.01). And the stability of conjugation antibodies on collagen films was significantly better than the control films.

CONCLUSIONChemical coupling is an effective approach to immobilize antibodies on collagen for further plasmid DNA tethering.

Angioplasty, Balloon, Coronary ; instrumentation ; Animals ; Antibodies, Antinuclear ; metabolism ; Cattle ; Coated Materials, Biocompatible ; chemistry ; metabolism ; Collagen ; chemistry ; metabolism ; Genetic Vectors ; Immunoglobulin M ; metabolism ; In Vitro Techniques ; Mice ; Protein Binding ; Stents ; Surface Properties

OBJECTIVETo evaluate the effect of antisense monocyte chemotactic protein-1 (A-MCP-1) nanoparticles (NPs) as gene carrier on gene transfer in two kinds of animal models.

METHODSPoly (lactic acid-co-glycolic acid) (PLGA) was used to make the NPs loaded with A-MCP-1 through a double-emulsion/solvent evaporation technique. NPs size was assessed by dynamic laser defractometer. The particle morphology was observed by scanning electron microscopy. DNA content in the NPs was measured by dissolving known amounts of NPs in chloroform and extracting DNA with water. In vitro release was performed in tris-EDTA buffer at 37 degrees C using double-chamber diffusion cells. The receiver buffer was replaced daily. The A-MCP-1 NPs was transfected into the cultured smooth muscle cells. PCR was used to evaluate the transfection of A-MCP-1. Cationic lipid (Lipofectamine) was used to transfect A-MCP-1 as control. After 48 hours incubation, cells were digested and examined by polymerase chain reaction. Twenty New Zealand white rabbits under jugular vein to artery bypass grafting procedure were divided into four groups: the first group received grafts treated with A-MCP-1 NPs, the second group received grafts treated with cationic liposome (dioleoyl trimethyl ammonium propane)-A-MCP-1, the third group received grafts treated with plasmid DNA, and the fourth group received grafts without transfection as control. Fourteen days after surgery the grafts were harvested. The expression of A-MCP-1 and its effect on MCP-1 in vein grafts were detected by dot blotting. The morphology of the grafts was investigated. To establish abdominal aortic aneurysms rats model, rats were randomly divided into three groups: A-MCP-1 NPs injection group, shame NPs injection group and control groups (without injection). Two weeks after surgery, diameter of abdominal aorta was measured and aortic tissue was obtained for PCR analysis to evaluate the A-MCP-1 expression. Western blot were applied to detect the inhibitory effect to the expression of MCP-1 mRNA and CD68 protein by A-MCP-1 NPs.

RESULTSNPs size ranged 198nm to 205nm with average around 201.4 nm. DNA content in the NPs was 4.14%. NPs showed steady release rate in vitro in Tris-EDTA solution. It released faster in the first week then maintained a slowly sustained release up to 16 days. In cell culture A-MCP-1 gene successfully transfected into smooth muscle cells by NPs vector. In vein grafting animal model, A-MCP-1 expression was detected in the vascular walls of NPs and cationic lipid treated groups. The degree of vascular hyperplasia in the gene NPs treated group was significantly lower than that in control group. There was no significant difference in the inhibition of intimal hyperplasia between NPs and cationic lipid treated groups. Two weeks after transfection in abdominal aortic aneurysm rats models, the abdominal aortic diameter of A-MCP-1 NPs injection group was (1.79 +/- 0.12) mm, significantly smaller than that of control groups [shame NPs group was (2.58 +/- 0.21) mm, and saline group was (2.63 +/- 0.29) mm] (P < 0.01). The expressions of MCP-1 mRNA and CD68 protein in A-MCP-1 NPs injection group were 12.5 +/- 1.5 and 17.6 +/- 2.1, which were much lower than those in control group [in shame NPs group, which were 35.7 +/- 4.5, 42.3 +/- 5.7 (P < 0.01), and saline group which is 32.4 +/- 3.9, 39.8 +/- 4.8 (P < 0.01)]. Specific band of A-MCP-1 was detected only in the A-MCP-1 NPs injection group by PCR.

CONCLUSIONA-MCP-1 gene NPs can be successfully used in rabbit vein grafting model and abdominal aortic aneurysm rats models, and may be potentially applied in clinical practice.

Animals ; Aortic Aneurysm ; genetics ; Chemokine CCL2 ; genetics ; metabolism ; Gene Transfer Techniques ; Genetic Vectors ; Lactic Acid ; chemistry ; Models, Animal ; Nanoparticles ; Oligonucleotides, Antisense ; genetics ; Polyglycolic Acid ; chemistry ; Polymers ; chemistry ; Rabbits ; Rats ; Transfection

OBJECTIVETo explore the possibility of using an endovascular microcoil as a gene delivery system.

METHODSAnti-adenoviral monoclonal antibodies were covalently attached to the collagen-coated surface of platinum microcoil. These antibodies were used to tether adenovirus encoding green fluorescent protein (Ad-GFP). Cell culture studies with rat arterial smooth muscle cells (A10) assessed transduction on or near the coil. Platinum coils coated with Ad-GFP were implanted into the ligated common carotid artery (CCA) of adult rats in a model of arterial stasis and pressurization. After 7 days, CCA segments were harvested, and coils were removed for histopathology and GFP expression studies, while organs were evaluated by polymerase chain reaction to assess viral biodistribution.

RESULTSIn cell culture studies, GFP-positive smooth muscle cells were detected only on the platinum coil surface. After 7 days, GFP was detected on the harvested platinum coil and in the organizing thrombus within the CCA according to fluorescence microscopy and immunohistochemistry. Morphometric analyses revealed that (13.3 +/- 2.0)% of cells within the organized thrombus were transduced with Ad-GFP via the gene delivery system. Ad-GFP was not detectable by polymerase chain reaction in lung, liver, or kidney.

CONCLUSIONSGene delivery endovascular microcoils represents an interventional device-based gene therapy system that can serve as a suitable platform for either single or multiple gene therapy vectors.

Adenoviridae ; genetics ; immunology ; Aneurysm ; surgery ; Animals ; Antibodies, Viral ; chemistry ; metabolism ; Biological Availability ; Carotid Artery, Common ; drug effects ; metabolism ; surgery ; Cells, Cultured ; Drug Delivery Systems ; instrumentation ; Embolization, Therapeutic ; methods ; Endothelial Growth Factors ; therapeutic use ; Genetic Therapy ; instrumentation ; methods ; Genetic Vectors ; administration & dosage ; chemistry ; Green Fluorescent Proteins ; analysis ; Muscle, Smooth, Vascular ; cytology ; Platinum ; chemistry ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; therapeutic use ; Transduction, Genetic ; instrumentation ; methods

OBJECTIVETo prepare a biodegradable drug-eluting stent in myocardium channel and evaluate its effect on myocardium channel after transmyocardial revascularization (TMR).

METHODSA biodegradable drug-eluting stent was prepared using poly (epsilon-caprolactone) (PCL), bovine serum albumin (BSA), and poly (D, L-lactide-co-glycolide) (PLGA) as material of stent, model protein drug, and drug carrier respectively. The amount of BSA in stent and in vitro released BSA of stent were determined by the Coomassie brilliant blue assay. The mechanical strength of stent was tested by universal material testing machines. The material and structure of stent was characterized by nuclear magnetic resonance spectroscopy. The effect of stent on myocardium channel after TMR was evaluated in vivo by a standard animal model of chronic myocardial ischemia in miniswine.

RESULTSThe stent could carry 13.1 microg BSA per mg of stent and the stent could release about 95% of BSA after 30 days. The stent diminished 80% of initial scale under the stress of 1.7 Mpa. It also kept the myocardium channel patency after TMR.

CONCLUSIONSA biodegradable drug-eluting stent in myocardium channel was successfully prepared. It can sustain the pressure from the heart and achieve the controlled release of drug. The stent can ensure the myocardium channel patency after TMR.

Animals ; Biocompatible Materials ; chemistry ; Blood Vessel Prosthesis ; Caproates ; chemistry ; Cardiac Surgical Procedures ; Disease Models, Animal ; Drug Delivery Systems ; instrumentation ; Heart ; drug effects ; Humans ; Lactones ; chemistry ; Myocardial Ischemia ; drug therapy ; surgery ; Myocardial Revascularization ; instrumentation ; Random Allocation ; Swine ; Swine, Miniature

AIMTo evaluate the effects of an array of additives on drug release from double-layered poly(lactic-co-glycolic acid) (PLGA) matrices.

METHODSAdditives differing in molecular size, hydrophilicity and steric configuration were selected for this study. An anti-proliferative 2-aminochromone, U-86983 (U-86, Pharmacia and Upjohn), was used as a model agent because of our interest in investigating local drug delivery systems for the inhibition of restenosis.

RESULTSIn vitro release of U-86 PLGA matrices without additive showed a typical biphasic release kinetics, i.e. a slow diffusion release (Phase I) followed by a fast erosion-mediated release (Phase II). The water-soluble additives in PLGA matrices changed the biphasic release pattern to a near monophasic profile by increasing the release of the Phase I. Increasing the ratio of additives to PLGA in matrices caused a significant increase in U-86 release rates. A high molecular weight water-soluble additive, Pluronic F127, resulted in a matrix showing perfect zero-order release kinetics. The morphologic evaluation of matrices using scanning electron microscopy indicated that the water-soluble additives were leachable and thus generated a highly porous structure in the matrices. Conclusion Water-solubility, molecular size and steric configuration of additives are the important determinants in generating various types of pore structures in polymer matrix which in turn affect the release mechanism and release kinetics.

Biocompatible Materials ; chemistry ; Chromones ; administration & dosage ; chemistry ; Delayed-Action Preparations ; Drug Carriers ; Drug Delivery Systems ; Excipients ; chemistry ; Lactic Acid ; chemistry ; Molecular Weight ; Morpholines ; administration & dosage ; chemistry ; Pharmacokinetics ; Poloxamer ; chemistry ; Polyglycolic Acid ; chemistry ; Polymers ; chemistry ; Tartrates ; chemistry

OBJECTIVETo experimentally investigate direct intramuscular gene transfer of nanoparticles encoding vascular endothelial growth factor for the treatment of peripheral artery disease.

METHODSThe human VEGF(165) cDNA was cloned into the eukaryotic expression vector PIRES2 under the control of cytomegalovirus promoter/enhancer. The recombinant gene was transferred into a rabbit model of chronic hindlimb ischemia by naked plasmid and nanoparticle respectively. Ischemia was induced in the hindlimb of New Zealand White rabbits by ligation of the distal external iliac artery and complete excision of the femoral artery and all its branches. At day 7 postoperation animals received VEGF(165) plasmid (10 intramuscular) or nanoparticle-VEGF(165) (8 intramuscular). With RT-PCR, immunohistochemistry analysis, and angiography, the expression and biological effects of VEGF(165) gene in experimental animals were investigated.

RESULTSTwo weeks after initiation of therapy, angiography showed that the transfer of VEGF(165) gene stimulated the formation of focal neovessels and established collateral circulation. The adductor muscle of ischemic limbs was histologically examined at day 14. Capillary density was increased among VEGF(165)-transfected rabbits, especially Nano-VEGF(165)-treated animals (Naked VEGF(165) plasmid = 50.18 per mm(2), Nano-VEGF(165) = 81.22 per mm(2), Control = 29.54 per mm(2), P < 0.05). RT-PCR showed that the transcription and expression of VEGF(165) gene in experimental group were significantly higher than those of control groups.

CONCLUSIONSIntramuscular administration of VEGF(165) induces collateral artery augmentation in the rabbit model of chronic limb ischemia. Nanoparticle can act as a vector to transfect specific gene and it will benefit gene transfer.

Angiography ; Animals ; Capsules ; Chronic Disease ; Disease Models, Animal ; Genetic Therapy ; methods ; Hindlimb ; blood supply ; Immunohistochemistry ; Ischemia ; genetics ; therapy ; Male ; Nanotechnology ; Particle Size ; Rabbits ; Reverse Transcriptase Polymerase Chain Reaction ; Treatment Outcome ; Vascular Endothelial Growth Factor A ; genetics ; therapeutic use

OBJECTIVETo explore the feasibility of utilizing two implantable devices made from modified polyurethane films with antibody tethered replication-defective adenoviruses encoding for green fluorescent protein (AdGFP) as gene delivery platforms.

METHODSIntra-aortic button implants of collagen-coated polyurethane films with antibody tethered AdGFP were sutured into the infrarenal aorta of adult pigs and pulmonary valve leaflet in juvenile sheep was replaced by polyurethane pulmonary valve cusp replacement with antibody-tethered AdGFP. After seven days, the buttons, prosthetic leaflets, and their surrounding tissues were explanted and evaluated for biocompatibility and AdGFP-mediated gene transfer by fluorescent microscopy and PCR analysis.

RESULTSIn vivo analysis of gene transfer from collagen-coated polyurethane films in pig infrarenal aorta implants, one week explants of the collagen-coated polyurethane films demonstrated (14.2 +/- 2.5)% of neointimal cells on the surface of the implant. In sheep pulmonary valve leaflet replacement studies, polyurethane films with antibody tethered AdGFP vector demonstrated (25.1 +/- 5.7)% of cells attached to polyurethane valve leaflets were transduced in one week. PCR analyses showed that GFP DNA was not detectable in blood or distal tissues.

CONCLUSIONSite-specific intravascular delivery of adenoviral vectors for gene therapy can be achieved with these two kinds of polyurethane implants utilizing the antivector antibody tethering mechanism.

Adenoviridae ; genetics ; Animals ; Blood Vessel Prosthesis ; Gene Transfer Techniques ; Genetic Therapy ; methods ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Heart Valve Prosthesis ; Male ; Polyurethanes ; chemistry ; Prosthesis Implantation ; Sheep ; Swine

OBJECTIVETo explore the feasibility of using an endovascular metal stent as a highly efficient and site-specific gene delivery system.

METHODSStents were formulated with a collagen coating. Anti-DNA monoclonal antibodies were covalently bound to the collagen surface by a cross linking reagent of N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). Binding capacity and stability of antibody and plasmid DNA on stents were quantified by radioactive labeling. The gene transduction efficiency was evaluated in cell culture and in rabbits.

RESULTSThe amount of antibodies binding on collagen matrix through SPDP reaction was 15 times higher than that of through physical absorption (P < 0.005). The binding stability of plasmid was significantly better than the control groups (P < 0.01). There was no harmful effect on cell growth with the anti-DNA antibody modified stents. The stents retrieved from cell culture after 72 hours of incubation in A10 cells showed numerous transducted cells only infiltrating the surface coating indicating a highly localized and efficient gene delivery pattern. Results of in vivo gene transfer by this modified stent revealed (2.8 +/- 0.7)% of total cells transduction and the higher transduction location was neointimal layer (about 7%). No distal spread of vector was detectable in the anti-DNA antibody modified stent implantation animals.

CONCLUSIONSAnti-DNA antibody modified stents represent a novel highly efficient and site-specific gene delivery system which can deliver various kinds of plasmid vectors. The release of plasmid DNA tethered on the stents could be controlled in some conditions. This novel system provided a novel platform for cardiovascular site-specific gene therapy.

Animals ; Antibodies, Antinuclear ; immunology ; Antibodies, Monoclonal ; immunology ; Cells, Cultured ; Coated Materials, Biocompatible ; Collagen ; DNA ; genetics ; Gene Transfer Techniques ; Genetic Vectors ; Male ; Mice ; Plasmids ; Rabbits ; Stainless Steel ; Stents

The novel paclitaxel-loaded nanopaticle through surface modification with didodecylmethylammonium bromide (DMAB) was prepared and its prevenative against neointimal formation in a rabbit carotid artery injury model was tested. Paclitaxel-loaded nanoparticles were prepared from oil-water emulsions using biodegradable poly (lactic acid-co-glycolic acid) (PLGA). Specific additive for surface conjugation was added after particle formation. To enhance arterial retention using a cationic surfactant, DMAB, was used. The size and distribution, surface morphology and surface charge of the paclitaxel-loaded nanoparticles were then investigated by laser light scattering, scanning electron microscope and zeta potential analyzer. The drug encapsulation efficiency (EE) and in vitro release profile were measured by high-performance liquid chromatography (HPLC). Balloon injured rabbit carotid arteries were treated with single infusion of the paclitaxel-loaded NP suspension and observed for 28 days. The inhibitory effects of vascular smooth muscle cell migration and proliferation were evaluated as end-point. The NPs showed spherical shape with diameter ranging from 200 to 500 nm. The negatively charged PLGA NPs shifted to positive after the DMAB modification. The in vitro drug release profile showed a triphasic release pattern. 28 days later, morphologic analysis revealed that the inhibitory effect of intima proliferation is dose-dependent, and the 30 mg x mL(-1) nanoparticle concentration suspension could completely inhibit proliferation of intima. Paclitaxel-loaded nanoparticles through surface modification with DMAB provide an effective means of inhibiting proliferation response to vascular injury in the rabbit.
Animals ; Antineoplastic Agents, Phytogenic ; administration & dosage ; chemistry ; therapeutic use ; Carotid Artery Injuries ; etiology ; pathology ; prevention & control ; Catheterization ; adverse effects ; Delayed-Action Preparations ; Drug Compounding ; Drug Delivery Systems ; Female ; Hyperplasia ; prevention & control ; Lactic Acid ; chemistry ; Male ; Nanoparticles ; Nanotechnology ; Paclitaxel ; administration & dosage ; chemistry ; therapeutic use ; Particle Size ; Polyglycolic Acid ; chemistry ; Rabbits ; Tunica Intima ; injuries ; pathology

OBJECTIVETo sought to engineer and characterize a biodegradable nanoparticles (NPs) containing rapamycin which use poly (lactic-co-glycolic) acid (PLGA) as the carrier matrix and to assess its in vivo release characteristics by local drug delivery system intravascularly.

METHODSRapamycin-loaded PLGA NPs were prepared by an emulsification/solvent evaporation technique, and NPs size distribution was assessed by submicro laser defractometer. The particle morphology was observed by scanning electron microscopy. In vitro release from the NPs was performed in TE buffer at 37 degrees C under rotation utilizing double-chamber diffusion cells on a shake stander. In vivo NPs intravascular local delivery were performed by DISPATCH catheter in New Zealand rabbit abdominal aorta and Chinese experimental mini-pigs coronary artery models.

RESULTSBiodegradable rapamycin loaded PLGA NPs were constructed successfully by emulsification solvent-evaporation technique. The diameter of rapamycin-PLGA NPs was around 246.8 nm with very narrow size distribution, and rapamycin-NPs showed good spherical shape with smooth uniform surface. Rapamycin loaded in NPs were around was 19.42%. Encapsulation efficiency of drug was over 77.53%. The in vitro release of rapamycin from NPs showed that 75% of the drug was sustained released over 2 weeks and controlled release in a linear pattern. After a single 10 minutes infusion of rapamycin-PLGA NPs suspension (5 mg/ml) under 20.27 kPa through DISPATCH catherter in vivo, the mean rapamycin levels at 7 day and 14 day were (2.438 +/- 0.439) and (0.529 +/- 0.144) microg/mg of the dry-weight of the artery segments (2 cm) which local delivery were administrated.

CONCLUSIONSPLGA NPs controlled drug delivery system for intraarterial local anti-proliferative drug delivery can potentially improve local drug concentration and prolong drug residence time in animal model in vivo. It should be appropriate for further study of its therapy efficiency in human.

Animals ; Aorta, Abdominal ; drug effects ; Coronary Vessels ; drug effects ; Drug Carriers ; chemistry ; Drug Delivery Systems ; methods ; Infusions, Intra-Arterial ; Lactic Acid ; chemistry ; Nanoparticles ; chemistry ; Particle Size ; Polyglycolic Acid ; chemistry ; Rabbits ; Sirolimus ; administration & dosage ; pharmacokinetics ; Swine ; Swine, Miniature