Cunxian Song

Formulation and characterization of biodegradable nanoparticles for intravascular local drug delivery

Abstract Various drug-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) were prepared using an emulsification/solvent evaporation technique. Different emulsion systems were employed according to the solubility of individual drugs so that an optimal drug incorporation efficiency and release profile were achieved for a variety of model compounds. Bovine serum albumin was studied as a model protein. Several specific Pharmacia and Upjohn drugs, U-86983, U-61431F, and U-74389G, as well as dexamethasone were tested because of our interest in intravascular drug delivery for the prevention of post-angioplasty restenosis. Drug loading in nanoparticles ranged from 10% to 30%. Typical particle size ranged from 60–200 nm with 85% of the particles in the range of 70–165 nm. The in vitro release rate for albumin was dependent upon the molecular weight (MW) of PLGA. Low MW (58 000) PLGA resulted in much faster BSA release than that of high MW (102 000) PLGA over 7 weeks. Cross-linking on the NP surface reduced the rate of drug release. Nanoparticle uptake by the arterial wall was evaluated by an ex vivo model utilizing freshly explanted dog carotid arteries. It was demonstrated that about 26% of the infused NP was initially retained by the intravascular matrix. A fraction (about 20%) of the initially retained NP remained in the arterial tissue 30 min or more after the end of the infusion. Nanoparticles with smaller mean size (100 nm vs. 266 nm) and lower drug loading (13.1% vs. 20.7%) resulted in higher arterial uptakes compared to nanoparticles of larger size and higher drug loadings. Sterilization of the drug-loaded nanoparticles by γ -irradiation at 2.5 Mrad dose showed no adverse effect on particle size, drug release behavior as well as ex vivo arterial uptake of the nanoparticles. In conclusion, this study demonstrated that a wide variety of water soluble and insoluble bioactive agents can be incorporated into PLGA nanoparticles with a high efficiency and adjustable drug loadings. By choosing the composition and the molecular weight of the polymeric matrix, the drug release kinetics from the nanoparticles can be controlled. Drug-loaded PLGA nanoparticles show great potential in intravascular local drug delivery.

Local intraluminal infusion of biodegradable polymeric nanoparticles: A novel approach for prolonged drug delivery after balloon angioplasty

BACKGROUND Several perfusion balloon catheters are under investigation for local drug delivery; however, sustained tissue drug levels are difficult to achieve with these techniques. To overcome this problem, sustained-release, biodegradable nanoparticles represent a potential alternative for prolonged local delivery. METHODS AND RESULTS A biodegradable polylactic-polyglycolic acid (PLGA) copolymer was used to formulate nanoparticles. Fluorescent-labeled nanoparticles were intraluminally administered in a single, 180-second infusion after balloon injury in the rat carotid model. Localization and retention at different time points and biocompatibility of nanoparticles were evaluated. To evaluate the potential of the system in the prevention of neointimal formation, dexamethasone was incorporated into the particles and delivered locally as above. Nanoparticles were seen in the three layers of the artery at 3 hours and 24 hours. At 3 days, they were mainly present in the adventitial layer, decreasing at 7 days, with no fluorescent activity at 14 days. The PLGA nanoparticles appeared to be fully biocompatible. In the dexamethasone nanoparticle study, a significant amount of dexamethasone was present in the treated segment for up to 14 days after a single infusion, with no plasma levels detected after the first 3 hours. There was a 31% reduction in intima-media ratio in animals treated with local dexamethasone nanoparticles compared with control. CONCLUSIONS Nanoparticles successfully penetrated into the vessel wall and persisted for up to 14 days after a short, single intraluminal infusion. Local administration of nanoparticles with incorporated dexamethasone significantly decreased neointimal formation. This methodology appears to have important potential for clinical applications in local drug delivery.

Arterial uptake of biodegradable nanoparticles for intravascular local drug delivery: Results with an acute dog model

Biodegradable nanoparticles (NP) with a spherical diameter ranging from 70 to 160 nm were investigated for potential usefulness for the local intraluminal therapy of restenosis, the disease process responsible for arterial reobstruction following angioplasty. NPs containing a water-insoluble anti-proliferative agent U-86983 (U-86, Pharmacia and Upjohn, Kalamazoo, MI) were formulated from oil-water emulsions using biodegradable polymers such as poly(lactic acid-co-glycolic acid) (PLGA), and specific additives after particle formation, to enhance arterial retention using either heparin, didodecylmethylammonium bromide (DMAB), or fibrinogen, or combinations. Femoral and carotid arteries of male mongrel dogs were isolated in situ, and were then subjected to a balloon angioplasty. A NP suspension of a predetermined concentration was then infused into the artery for various durations. This was followed by a 30 min restoration of blood flow through the vessel. The arterial segments were excised and analyzed for drug levels. From the drug loading the NP and the drug levels in the artery, the quantity of nanoparticles retained was calculated and expressed as microgram per 10 mg dry arteries. In general, repeated short infusions of nanoparticle suspension (15 s x 4) were two-fold more effective in terms of higher arterial U-86 levels than a single prolonged infusion (60 s). A single 15 s infusion was not significantly different than a 60 s compared to non-modified NPs (39.2 +/- 2.5 and 49.1 +/- 2.4 vs. 21.5 +/- 0.6 micrograms/10 mg mean +/- s.e., respectively). A comparably enhanced NP uptake was noted with a combined heparin/DMAB modification. Increasing the concentration of NP in infusate from 5 to 30 mg ml-1 significantly increased arterial NP uptake level (from 22.5 +/- 3.5 to 83.7 +/- 1.4 micrograms/10 mg). Thus, the results support the view that modified nanoparticles along with optimized infusion conditions could enhance arterial wall drug concentrations of agents to treat restenosis.

Nanoparticle drug delivery system for restenosis

Abstract Restenosis, an arterial reobstruction occurring in thirty to fifty percent of patients undergoing coronary angioplasty, because of its localized nature can possibly be best treated by local drug therapy. In recent years, several local drug delivery strategies have been investigated for the prevention of restenosis. In this review we discuss the therapeutic potential of nanoparticles as a drug delivery system for restenosis. Nanoparticles, for the purpose of this review, are 30–500 nm diameter polymeric spherical particles. Nanoparticles possess several advantages as a carrier system for the intra-arterial localization of therapeutic agents. These advantages include their subcellular size, targeted surfaces, good suspensibility, and uniform dispersity for a catheter-based delivery, and an easy penetration into the arterial wall without causing trauma. In various studies we have demonstrated an efficient intra-arterial localization of nanoparticles, biocompatibility in the arterial wall, and effectiveness for the inhibition of experimental restenosis. Thus, nanoparticles could prove to be an effective dosage form for an intra-arterial localization of therapeutic agents for preventing restenosis in a clinical setting.

Gene Delivery to Pig Coronary Arteries from Stents Carrying Antibody-Tethered Adenovirus

Deployment of coronary stents to relieve atherosclerotic obstruction has benefitted millions of patients. However, gene therapy to prevent in-stent restenosis, while promising in experimental studies, remains a challenge. Conventional strategies for viral vector administration utilize catheters that deliver infusions of viral suspensions, which result in suboptimal localization and potentially dangerous distal spread of vector. Stent-based gene delivery may circumvent this problem. We hypothesized that site-specific delivery of adenoviral gene vectors from a stent could be achieved through a mechanism involving anti-viral antibody tethering. Stents were formulated with a collagen coating. Anti-adenoviral monoclonal antibodies were covalently bound to the collagen surface. These antibodies enabled tethering of replication defective adenoviruses through highly specific antigen-antibody affinity. We report for the first time successful stent-based gene delivery using antibody-tethered adenovirus encoding green fluorescent protein (GFP), demonstrating efficient and highly localized gene delivery to arterial smooth muscle cells in both cell culture and pig coronary arteries. Overall arterial wall transduction efficiency in pigs was 5.9 +/- 1.1% of total cells. However, neointimal transduction was more than 17% of total cells in this region. Importantly, when specific antibody was used to tether adenovirus, no distal spread of vector was detectable by PCR, in either distal organs, or in the downstream segments of the stented arteries. Control adenovirus stents, with nonspecific antibody plus adenovirus, demonstrated only a few isolated foci of transduction, and poor site-specific transduction with distal spread of vector. We conclude that a vascular stent is a suitable platform for a localizable viral vector delivery system that also prevents systemic spread of vector. Gene delivery using stent-based anti-viral antibody tethering of vectors should be suitable for a wide array of single or multiple therapeutic gene strategies.

Controlled release of U-86983 from double-layer biodegradable matrices: Effect of additives on release mechanism and kinetics

Abstract Effects of additives on the drug release kinetics from biodegradable matrices is an important determinant in designing a drug delivery system. These experiments introduced the influence of an array of additives on the drug release from double-layered poly(lactic-co-glycolic acid) (PLGA) matrices. Various additives such as l -tartaric acid dimethyl ester (DMT), Pluronic® F127 (F127); 2-hydroxypropyl derivative of β-cyclodextrin (HPB), methyl derivative of β-cyclodextrin (MMB) and Beeswax (Wax), differing in molecular size, hydrophilicity and steric configuration were selected for this study. An antiproliferative 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. The in vitro release of U-86 from 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 rate of the Phase I. Increasing the ratio of additives to PLGA in matrices causes a significant increase in the U-86 release rates. The high molecular weight water-soluble additive, Pluronic® F127, resulted in a matrix showing perfect zero-order release kinetics. The water-soluble cyclodextrin derivative, HPB, gave the highest release rate among all the matrices formulated. A hydrophobic additive, Beeswax, however showed biphasic release kinetics comparable to PLGA control matrices, but delayed the onset of the Phase II by 4 days. The U-86 release profiles were in good agreement with the mass loss profiles of these matrices except for the matrices with F127 and HPB additives. The morphologic evaluation of matrices using scanning electron microscopy indicates that the water-soluble additives are leachable and thus generate a highly porous structure in the matrices. The matrix pore configuration (e.g. interconnected or closed) created with different additives determined the mechanism of drug release kinetics from the various matrix formulations. In conclusion, the feasibility of modulating release rates and kinetics of an agent from PLGA monolithic matrices by utilizing various types of additives is demonstrated. Water-solubility, molecular size and steric configuration of the 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.

The Incorporation of an Ion Channel Gene Mutation Associated with the Long QT Syndrome (Q9E-hMiRP1) in a Plasmid Vector for Site-Specific Arrhythmia Gene Therapy: In Vitro and In Vivo Feasibility Studies

The present studies investigated the cardiac potassium channel missense mutation, Q9E-hMiRP1, for potential use as a gene therapy construct for cardiac arrhythmias. This gene abnormality is one of a number of mutations that can cause the long QT syndrome (LQTS), a hereditary arrhythmia disorder that is associated with sudden death. However, individuals who carry the Q9E-hMiRP1 variant are predisposed to developing the LQTS only after clarithromycin administration. Because the electrophysiologic mechanism of action of Q9E-hMiRP1 (i.e., diminished potassium currents resulting in delayed myocardial repolarization) is comparable to that of class III antiarrhythmic agents, we examined Q9E-hMiRP1 as a candidate gene therapy construct for site-specific treatment of reentrant atrial cardiac arrhythmias. Our rationale was also based on the hypothetical safety of the atrial use of Q9E-hMiRP1 because LQTS characteristically causes ventricular but not atrial arrhythmias. Furthermore, the possible use of clarithromycin to control the conduction effects of overexpressed Q9E-hMiRP1 pharmacologically was another attractive feature. In our studies we investigated the use of two bicistronic plasmid DNA gene vectors with either hMiRP1 or Q9E-MiRP1 and green fluorescent protein (GFP), plus a C-terminus of the hMiRP1 or of the Q9E-hMiRP1 coding region for the FLAG (MDYKDDDDK) peptide. We generated two stable cell lines using HEK293 and SH-SY5Y (human cell lines), overexpressing the genes of interest, confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blots. The expected plasma membrane localization of each overexpressed transgene was confirmed by immunofluorescent confocal fluorescent microscopy using anti-FLAG antibody. Patchclamp studies demonstrated that cells transfected with Q9E-hMiRP1 plasmid DNA exhibited significantly reduced potassium currents but only with clarithromycin administration. A novel plasmid DNA delivery system was formulated for use in our animal studies of the hMiRP1 vectors, which was composed of DNA-anti-DNA antibody-cationic lipid (DAC) heteroplexes. In vitro and in vivo studies using DAC heteroplexes containing anti-DNA antibodies with nuclear targeting capability demonstrated significantly increased transfection compared to naked DNA, and to DNA-cationic lipid complexes. Pig atrial myocardial injections of DAC heteroplexes demonstrated 16% of regional cardiac myocytes transfected using the Q9E-hMiRP1 plasmid, and 15% of cells with the hMiRP1 vector. It is concluded that the present studies support the view that site-specific gene therapy for atrial arrhythmias is feasible using plasmid vectors for overexpressing ion channel mutations that have electrophysiologic effects comparable to class III antiarrhythmic agents.

The effect of intramural delivery of polymeric nanoparticles loaded with the antiproliferative 2-aminochromone U-86983 on neointimal hyperplasia development in balloon-injured porcine coronary arteries

Abstract Applying local drug delivery techniques in treating restenosis with antiproliferative agents may prove useful in enhancing drug efficacy while minimizing inherent systemic toxicity. Using direct intramural delivery of drug-loaded nanoparticles (U-86NP) composed of the biodegradable co-polymer polylactic-polyglycolic acid (PLGA), we evaluated the in vivo antiproliferative effect of a novel 2-aminochromone, U-86983, in a porcine model of coronary artery neointimal hyperplasia. In vitro, PLGA nanoparticles proved a suitable carrier for U-86983, promoting the gradual release of biologically active drug over a 2-week period. A series of acute canine and porcine experiments subsequently clarified such delivery conditions as catheter device, nanoparticle surface modification, and suspension concentration that maximized U-86NP retention in balloon-injured arterial segments. A chronic efficacy study implementing these delivery conditions was then performed in domestic feeder pigs. Site-specific delivery of U-86NP significantly reduced neointimal hyperplasia in severely balloon-injured porcine coronary arteries but also increased the incidence of medial dissection at the treated site. Delivery conditions designed to maximize drug effects without exacerbating existing vascular injury remain to be elucidated.

Polymeric drug delivery systems for treatment of cardiovascular calcification, arrhythmias and restenosis

Abstract Cardiovascular controlled release, utilizing drug-polymer composites implanted in direct contact with the heart, has recently come into clinical use with a dexamethasone eluting cardiac pacemaker lead tip. Furthermore, cardiovascular controlled release systems are under active investigation in a number of other areas of possible application. The general working hypothesis of this approach is that regionally administered drug delivered directly to the heart or a blood vessel will more efficiently and effectively treat localized disease processes of interest, while avoiding systemic side effects. Successful experimental examples illustrating the validity of this hypothesis have involved investigations into cardiovascular calcification, therapy of cardiac arrhythmias, and treatment of arterial restenosis following angioplasty. Efficacious results in each of these areas, with some limitations, have been noted, and are discussed in detail in this paper. An ideal cardiovascular controlled release system will consist of a feedback responsive implant in which drug release kinetics could be varied according to disease activity, or other considerations such as side effects of the therapeutic agent. Furthermore, cardiovascular drug delivery should be ideally extremely long acting and this may be possible through the use of therapeutic agents in a refillable reservoir configuration, or local gene therapy with long standing expression of the gene of interest.

Synthesis, characterization, and evaluation of paclitaxel loaded in six-arm star-shaped poly(lactic-co-glycolic acid)

Background Star-shaped polymers provide more terminal groups, and are promising for application in drug-delivery systems. Methods A new series of six-arm star-shaped poly(lactic-co-glycolic acid) (6-s-PLGA) was synthesized by ring-opening polymerization. The structure and properties of the 6-s-PLGA were characterized by carbon-13 nuclear magnetic resonance spectroscopy, infrared spectroscopy, gel permeation chromatography, and differential scanning calorimetry. Then, paclitaxel-loaded six-arm star-shaped poly(lactic-co-glycolic acid) nanoparticles (6-s-PLGA-PTX-NPs) were prepared under the conditions optimized by the orthogonal testing. High-performance liquid chromatography was used to analyze the nanoparticles’ encapsulation efficiency and drug-loading capacity, dynamic light scattering was used to determine their size and size distribution, and transmission electron microscopy was used to evaluate their morphology. The release performance of the 6-s-PLGA-PTX-NPs in vitro and the cytostatic effect of 6-s-PLGA-PTX-NPs were investigated in comparison with paclitaxel-loaded linear poly(lactic-co-glycolic acid) nanoparticles (L-PLGA-PTX-NPs). Results The results of carbon-13 nuclear magnetic resonance spectroscopy and infrared spectroscopy suggest that the polymerization was successfully initiated by inositol and confirm the structure of 6-s-PLGA. The molecular weights of a series of 6-s-PLGAs had a ratio corresponding to the molar ratio of raw materials to initiator. Differential scanning calorimetry revealed that the 6-s-PLGA had a low glass transition temperature of 40°C–50°C. The 6-s-PLGA-PTX-NPs were monodispersed with an average diameter of 240.4±6.9 nm in water, which was further confirmed by transmission electron microscopy. The encapsulation efficiency of the 6-s-PLGA-PTX-NPs was higher than that of the L-PLGA-PTX-NPs. In terms of the in vitro release of nanoparticles, paclitaxel (PTX) was released more slowly and more steadily from 6-s-PLGA than from linear poly(lactic-co-glycolic acid). In the cytostatic study, the 6-s-PLGA-PTX-NPs and L-PLGA-PTX-NPs were found to have a similar antiproliferative effect, which indicates durable efficacy due to the slower release of the PTX when loaded in 6-s-PLGA. Conclusion The results suggest that 6-s-PLGA may be promising for application in PTX delivery to enhance sustained antiproliferative therapy.