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(Circulation. 1996;94:1441-1448.)
© 1996 American Heart Association, Inc.

Articles

Local Intraluminal Infusion of Biodegradable Polymeric Nanoparticles

A Novel Approach for Prolonged Drug Delivery After Balloon Angioplasty

Luis A. Guzman, MD; Vinod Labhasetwar, PhD; Cunxian Song, PhD; Yangsoo Jang, MD, PhD; A. Michael Lincoff, MD; Robert Levy, MD; Eric J. Topol, MD

the Department of Cardiology, Center for Thrombosis and Vascular Biology, the Cleveland Clinic Foundation, Cleveland, Ohio (L.A.G., Y.J., A.M.L., E.J.T.), and the Division of Pediatric Cardiology, the University of Michigan Medical School, Ann Arbor (V.L., C.S., R.L.).

Correspondence to Eric J. Topol, MD, Department of Cardiology, The Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195-5066.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
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.

Key Words: nanoparticles • arteries • balloons • angioplasty • drugs

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Despite extensive investigation during the past 15 years, restenosis in at least 30% to 50% of cases remains the major limiting factor in the long-term efficacy of percutaneous coronary balloon angioplasty (PTCA) and the related nonsurgical coronary revascularization procedures.^{1 2} Pharmacological therapy to decrease restenosis has shown promising results when tested in different animal models.^{1 2 3 4 5 6 7 8 9} However, clinical trials with these agents for the most part have failed to demonstrate any reduction in the restenosis rate.^{3 4 5 6 7 8 9} A possible explanation for these disappointing results may be related to differences in the drug dose administered or the difficulty in providing sustained administration of the agent to the target site.

Local drug delivery rather than systemic administration might be a more effective way to obtain higher tissue drug levels at the site of the balloon injury and at the same time decrease the potential adverse systemic drug-associated side effects. Our lab and others using the periadventitial drug delivery system have recently shown that via local delivery, drugs can markedly decrease the degree of neointimal proliferation without having significant systemic effects.^{10 11 12} However, the periadventitial delivery approach has little clinical applicability in the prevention of the restenosis process after PTCA.

Several local drug delivery systems, including different perfusion balloon catheters, hydrogel-coated balloon catheters, polymeric or coated stents, and a number of other approaches are currently under investigation.^{13 14 15 16} Recently, the first local delivery balloon catheters (the Dispatch delivery catheter, SciMed Inc, and LocalMed sleeve, Localmed, Inc) have been approved for intracoronary drug infusion by the US Food and Drug Administration. However, the main disadvantages of local catheter injection systems are the very low tissue uptake of the infused agents and the lack of sustained delivery with rapid washout of the agent over minutes to hours.^{17 18 19 20}

For these reasons, we have investigated a nanoparticulate sustained-release carrier system that can incorporate a significant amount of drug, is small enough in size to be delivered through the currently available balloon delivery catheters, and is able to continuously release the agent over a prolonged period of time. To formulate this carrier, a biodegradable polymeric material, polylactic-polyglycolic acid (PLGA) copolymer, shown to be safe in the human body, was used.^{21 22} With the use of this material, nanoparticles (mean size, 165±39 nm) were formulated and characterized.

To evaluate the feasibility, kinetics, and durability of local delivery of the nanoparticles after a single local infusion, we used the rat carotid injury model. In a second set of experiments, we tested the feasibility of using sustained-release nanoparticles loaded with dexamethasone to decrease neointimal formation after vascular injury in the same animal model. Dexamethasone was selected because of our previous experience using a periadventitial delivery system in which this agent significantly decreased neointimal formation in the rat carotid injury model.¹⁰

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Experimental Design
All animal experiments were performed according to the animal welfare policy of the American Heart Association and the Cleveland Clinic Foundation. The experimental protocol was approved by the Cleveland Clinic Foundation Animal Research Committee. In Fig 1, the experimental design is summarized. The study consisted of two substudies: study protocols 1 and 2. In study protocol 1, the localization, retention, and tissue biocompatibility of the nanoparticles were evaluated (Fig 1A). Fluorescent-labeled (rhodamine B) nanoparticles were used to determine the tissue localization and persistence of nanoparticles in the tissue after a single local infusion. Carotid balloon injury was induced in 10 male Sprague-Dawley rats weighing 300 to 350 g. After injury, 10 mg of fluorescent-labeled PLGA nanoparticles were mixed with 200 µL of saline and infused over 3 minutes (three 60-second infusions, with a 60-second period in between) at 2 atm of pressure in the left common carotid artery. The animals were killed after 3 hours, 24 hours, 3 days, 7 days, and 14 days (2 animals at each time point). To evaluate the biocompatibility of the nanoparticles in the vascular tissue, carotid injury was created in three groups of rats. After injury, one group received 200 µL of saline containing 10 mg of nanoparticles infused (as above) over 3 minutes at 2 atm of pressure; in a second group (sham control), no local delivery was carried out after injury; in a third group, 200 µL of saline without nanoparticles was locally infused over 3 minutes at 2 atm of pressure. The animals were killed 21 days later for histological and morphometric analysis.

View larger version (39K): [in this window] [in a new window]   Figure 1. Flow diagram of the study protocol. A, Study protocol 1. Common carotid injury was induced in 37 Sprague-Dawley rats, followed by local infusion of fluorescent-labeled nanoparticles (NP). The animals were killed at different time points for tissue detection and localization of nanoparticles. In another set of experiments, the tissue biocompatibility was evaluated to compare the effect of animals treated with nanoparticles versus animals treated with the vehicle or sham operation. B, Study protocol 2. Carotid injury was induced in 38 animals followed by local infusion of nanoparticles loaded with dexamethasone (Dx-NP). The animals were killed at different time points for plasma and tissue drug level analysis. In a second set of experiments, the response of intimal hyperplasia after vascular injury to locally delivered dexamethasone nanoparticles was evaluated and compared with local nondrug nanoparticles and intraperitoneally injected dexamethasone nanoparticles.

In study protocol 2, nanoparticles were loaded with dexamethasone (13% by weight) and locally delivered after injury to determine whether this local delivery system could inhibit neointimal growth in the rat carotid injury model (Fig 1B). Two groups of animals received 10 mg of dexamethasone nanoparticles, one locally and one intraperitoneally. A third group received 10 mg of nanoparticles (no dexamethasone) locally. The animals were killed 21 days after injury for morphometric evaluation. Another group of 8 animals were injured and then treated with a local infusion of dexamethasone nanoparticles. The animals were killed 1, 3, 7, and 14 days later for determination of tissue and plasma dexamethasone levels (2 animals at each time point).

Nanoparticle Formulation and Characterization
The nanoparticles were formulated by collaborating investigators at the University of Michigan (V.L., C.S., and R.L.). The PLGA nanoparticle procedure utilized an emulsification solvent evaporation technique (patent pending). In brief, 600 mg of PLGA was dissolved in 24 mL of methylene chloride. The organic phase was emulsified into 120 mL of an aqueous phase containing 2.5% polyvinyl alcohol (30 to 70 K molecular weight) by sonication over an ice bath for 10 minutes with the use of a probe-type sonicator with an energy output set at 55 W to form an oil-in-water emulsion. The oil-in-water emulsion thus formed was further stirred at room temperature over a magnetic stir plate for 18 hours to evaporate the organic solvents. Nanoparticles formed in this fashion were recovered by ultracentrifugation at 145 000g, washed three times with water to remove polyvinyl alcohol, resuspended, and lyophilized. For tissue localization studies, PLGA nanoparticles were fluorescent-labeled with rhodamine B (Sigma). Rhodamine B (4 mg) was dissolved in the initial step into the organic phase, followed by the same sequence as above. To formulate nanoparticles loaded with dexamethasone, the drug (200 mg) was added in the initial step of the nanoparticle formulation. Dexamethasone was dissolved in 4 mL acetone and PLGA (600 mg) in 24 mL methylene chloride. Both solutions were mixed to form an organic phase. The subsequent steps were the same as described previously for nanoparticle formulation. Nanoparticles were characterized for particle size with the use of a laser defractometer (NICOMP, model 370). The particle size ranged from 90 to 250 nm, with 76% of the particles in the range of 121 to 186 nm, with a mean size of 165±39 nm. Scanning electron microscopy also demonstrated uniform particle size distribution.

In Vitro Controlled Dexamethasone Release
In vitro dexamethasone release from the particles was assessed with the use of a double diffusion chamber separated by a Millipore membrane (0.1 µm, type SV). The donor side contained a 5-mL suspension of nanoparticles (1 mg/mL) in PBS (pH 7.3, 154 mmol/L), and the receiver side contained plain phosphate buffer. The receiver side buffer was replaced periodically with fresh buffer. The drug that diffused into the receiver side was quantified by high-pressure liquid chromatography.

Balloon Injury Technique and Local Nanoparticle Delivery
General anesthesia was induced with a mixture of ketamine (80 mg/kg) and xylazine (5 mg/kg) given intraperitoneally. Through a midline neck incision the left common, external, and internal carotid arteries were exposed by blunt dissection. A 2F Fogarty balloon catheter was introduced in the left external carotid artery via an arteriotomy and advanced to the origin of the left common carotid artery. The balloon was inflated sufficiently to generate slight resistance and withdrawn three times to consistently produce endothelial denudation of the entire length of the left common carotid artery. Upon removal of the balloon catheter, a PE 10 catheter was inserted into the left common carotid artery. The mid and distal portions of the left common carotid artery and the left internal carotid artery were temporarily tied off. This created a closed system that allowed contact of the nanoparticles only with the distal 15 mm of the left common carotid artery. Ten milligrams of PLGA nanoparticles was mixed with 200 µL of saline and sonicated on ice for 1 minute before delivery. The 200 µL of nanoparticle suspension was infused over 3 minutes at 2 atm of pressure. Three 1-minute infusions of 70 µL of the suspension, with a 1-minute period between infusions, were performed. After the nanoparticles were delivered, the ties were removed and the flow was restored. The left external carotid artery was ligated with 3.0 silk, and the skin was closed with wound clips.

Histological Analysis
For the first set of experiments in which tissue uptake was evaluated, the animals were killed at different time points. Rats were anesthetized with a mixture of ketamine (80 mg/kg) and xylazine (5 mg/kg) given intraperitoneally. Via a midline abdominal incision, the distal abdominal aorta was exposed and an 18-gauge intravenous catheter introduced above the aortic bifurcation. The animals received a lethal dose of sodium pentobarbital. Both jugular veins were excised, and saline was infused into the abdominal aorta to clear the intravascular system. The entire right and left carotid arteries were retrieved and immediately sectioned every 2 to 3 mm. Three consecutive segments from the left common carotid artery were immediately immersed in OCT compound (International Equipment Co) and stored in darkness at -70°C until histological evaluation. The blocks were frozen-sectioned with the use of a Tissue Tek II Cryostat (Miles Laboratories Inc), and the sections were evaluated under fluorescent microscopy with the use of a green light filter. The contralateral untreated right common carotid arteries from two animals also were sectioned every 2 to 3 mm and used as control segments.

For histological and morphometric analysis, after the intravascular system was cleared, pressure fixation was performed with the use of 10% formaldehyde solution infused over 5 minutes at 120 mm Hg. After perfusion fixation, the right and left carotid arteries, including the aortic arch, were retrieved and immersed in the same fixative until sectioning. The left common carotid arteries were sectioned every 3 mm from the proximal to the distal ends. These sections were embedded in paraffin for sectioning, and duplicate slides were stained with hematoxylin-eosin and Lawson's elastic–van Gieson. Three different segments, with the maximal neointimal proliferation of the left common carotid within the distal 15 mm, were selected for histological analysis.

Dexamethasone Tissue and Plasma Level Analyses
Tissue and plasma dexamethasone levels were measured by immunoassay at Endocrine Sciences, Calabasas Hill, Calif. In brief, plasma samples were extracted with hexane/ethyl acetate, then analyzed overnight in a Bush descending-portion paper chromatography system. Recovery through the procedure was monitored in each sample with ³H-dexamethasone. After incubation, separation of the bound and free fraction was achieved with an ammonium sulfate fractionation. ³H-dexamethasone in the free fraction was quantitated with the use of scintillation counting. The concentration of dexamethasone in each sample was determined with the use of a standard curve.

Tissue levels were measured in a similar fashion as plasma levels after extraction. Each tissue sample was finely cut into small pieces with scissors, transferred to a homogenization tube, and ground first with 2 mL of extraction solvent I (chloroform/ethanol, 1.4:1 vol/vol) with the use of a Teflon paddle driven STIR-PAN laboratory mixer at a speed of 2000 to 2500 rpm in a 37°C to 38°C water bath for 10 to 15 minutes. Two more extractions were done with extraction solvent II (chloroform/ethanol, 2:1 vol/vol) as described above. All the extracts were collected and centrifuged at 1000 rpm to remove particulate matter, and the supernatant was evaporated to dryness with the use of a Speed Vac concentrator (Savant Instruments, Inc) at 50°C for 2 hours. The arterial tissue after extraction was lyophilized for 48 hours, and the dry weight was recorded.

Data and Statistical Analyses
Morphometric analysis was performed by an observer blinded to the drug regimen using a computerized digital microscopic planimetry algorithm (Bioquant program). Cross-sectional areas of media, intima, and lumen were measured.

All data are presented as mean±SEM. Differences between the three groups were analyzed with the use of ANOVA with Scheffe's test for post hoc comparisons. For differences between two groups a two-tailed, unpaired Student's t test was used. Statistical significance was defined as P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Fluorescent Nanoparticle Tissue Uptake and Localization
Fig 2 (A through D) shows the tissue uptake and localization of rhodamine-labeled nanoparticles at different time points after a single local infusion. Three hours after infusion, nanoparticles were observed to be accumulated principally at the luminal surface of the artery but with fluorescent activity also noteworthy in the adventitial layer (Fig 2A). At 24 hours, the presence of the nanoparticles was more substantial in the adventitial layer, although nanoparticles were still present at the luminal surface and, in some segments, in the innermost portion of the media (Fig 2B). During these early time points, the fluorescent activity was present in all the segments and in most of the arterial circumferences. However, areas with more activity and areas with less or no activity were noted. Three days after infusion, nanoparticles were still present in the vessel wall, although the fluorescent activity was only present in the adventitial layer in the form of clusters in some sections, with no activity in the media or intima (Fig 2C). Not all the segments showed fluorescent activity, nor was the activity uniformly distributed in the entire circumference of the vessel at this time point. At all time points the particles were mainly seen in the outer adventitial layer, an area that corresponds to the location of the majority of the vasa vasorum. At 7 days, the fluorescent activity significantly decreased, with a few scattered clusters in the adventitial layer and no activity in the other layers of the artery. No fluorescent activity was observed 14 days after infusion.

View larger version (338K): [in this window] [in a new window]   Figure 2. Color microphotographs of histological sections from injured left common carotid arteries showing the tissue localization and tissue persistence of nanoparticles after a single endoluminal injection. A, Fluorescence microscopy of rhodamine B–labeled nanoparticles 3 hours after infusion showing nanoparticles in the medial luminal layer as well as in the adventitial layer (arrows) (magnification x100). B, Fluorescence microscopy of rhodamine B–labeled nanoparticles 24 hours after injection showing media and adventitial localization of nanoparticles (magnification x200). C, Fluorescence microscopy of rhodamine B–labeled nanoparticles 3 days after injection showing that the fluorescent activity persisted but only in the adventitial layer (arrow) (magnification x200). D, Fluorescence microscopy of a carotid artery 24 hours after vascular injury without rhodamine B–labeled nanoparticle treatment (control artery) (magnification x200).

Nanoparticle Tissue Compatibility
No differences in vascular or perivascular injury was noted in any of the three study groups. There were scattered leukocytes attached to the luminal surface of the vessel at 1 and 3 days after injury and nanoparticles infusion, but no significant accumulation was noted. Fig 3 (A and B) shows the morphometric results in the three groups of animals 21 days after vascular injury. The cross-sectional areas of lumen, intima, and media were similar in the three groups. The intima/media ratio was also very similar in the three experimental groups (nanoparticle group, 1.17±0.07; sham control, 1.2±0.05; local vehicle, 1.13±0.05; P=.75). There was no apparent inflammatory response in any of the three groups at 21 days, with no leukocyte accumulation at the injured site exposed to local infusion of nanoparticles.

View larger version (78K): [in this window] [in a new window]   Figure 3. Bar graphs summarizing morphometric results of the tissue compatibility study. A, Cross-sectional areas (mm2) of lumen, intima, and media 21 days after balloon injury in the three experimental groups. B, Intima/media ratio 21 days after balloon injury in the three treatment groups. No significant differences were found between the groups, suggesting biocompatibility between the nanoparticles (NP) and the vessel wall.

In Vitro Dexamethasone Nanoparticle Release
The dexamethasone release curve showed an initial burst phase during the first 36 to 48 hours, releasing 50% of the loaded drug (Fig 4). This initial phase was followed by a drug release at exponentially decreasing rates based on a cumulative percentage of released drug over a 4-week period. Approximately 85% of the loaded dexamethasone was released in 3 weeks.

View larger version (11K): [in this window] [in a new window]   Figure 4. Graph showing cumulative dexamethasone (13% wt/wt loaded) release as a percentage of the initial polymer loading from polylactic-polyglycolic acid nanoparticles over a 4-week period. Each data point represents the mean of two determinations.

Dexamethasone Plasma and Tissue Levels
The dexamethasone plasma and tissue levels at different time points after a single local infusion of 10 mg of 13% loaded dexamethasone nanoparticles are shown in the Table. A significant amount of dexamethasone was detected in plasma in the first 3 hours after administration. No dexamethasone plasma levels were detected at 3, 7, and 14 days after the single local infusion of dexamethasone nanoparticles. Tissue levels of dexamethasone were significantly higher in the locally treated segment with dexamethasone nanoparticles than in the adjacent segment and contralateral segment at every time point after infusion. This difference in tissue dexamethasone levels ranged from 45-fold higher than the contralateral segment at 3 hours after infusion to 277-fold higher at 3 days. After 3 days, no dexamethasone was detected either in the adjacent segment or in the contralateral segment; however, a detectable quantity of drug was still present in the treated segment for up to 14 days after a single 3-minute infusion. This proportional difference was consistent within the animals between the different segments evaluated at different time points. However, a significant variability between animals in the amount of dexamethasone measured in the tissue at the same time point was noted. Based on the in vitro release studies, each animal is estimated to have received a maximal dose of 1.1 mg of dexamethasone during the 3-week study period or on average 0.17 ng/kg per day.

View this table: [in this window] [in a new window]   Table 1. Dexamethasone Plasma and Tissue Concentration

Effect of Locally Delivered Dexamethasone Nanoparticles on Neointimal Formation
Fig 5 (A and B) shows the morphometric results in the three groups of animals. There was a significantly larger lumen cross-sectional area and smaller intima cross-sectional area in the arteries treated with local dexamethasone nanoparticles compared with intraperitoneally delivered dexamethasone nanoparticles and control arteries. There was no significant effect on intimal formation in animals treated with intraperitoneal dexamethasone nanoparticles compared with the control. There was a 31% reduction in intima/media ratio in animals treated with local dexamethasone nanoparticles compared with intraperitoneally infused or control animals (local dexamethasone nanoparticles group, 0.81±0.07; intraperitoneal dexamethasone nanoparticles group, 1.15±0.09; local nondrug nanoparticles group, 1.17±0.07; P<.006). The media cross-sectional area was similar in the three treatment groups (local dexamethasone nanoparticles group, 0.135±0.003 mm²; intraperitoneal dexamethasone nanoparticles group, 0.13±0.002 mm²; local nondrug nanoparticles group, 0.14±0.004 mm²; P=.43).

View larger version (49K): [in this window] [in a new window]   Figure 5. Bar graphs summarizing the morphometric results of local dexamethasone nanoparticles. A, Cross-sectional areas (mm2) of lumen, intima, and media 21 days after balloon injury in the three study groups. B, Intima/media ratio 21 days after balloon injury in the three study groups. A significant difference between groups was found, showing that locally delivered dexamethasone nanoparticles decrease the degree of neointimal response after vascular injury. NP indicates nanoparticles; Ip, intraperitoneal; and Dx-NP, dexamethasone-loaded nanoparticles.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study shows that a brief, single intraluminal delivery of nanoparticles at low pressure during a short period of time can achieve a significant uptake in the vessel wall, with persistence for at least 7 days. In addition, we have found that the adventitia of the vessel wall was the principal reservoir of nanoparticles after the first 24 hours. The PLGA nanoparticles are biocompatible with the rat vascular tissue, as demonstrated by the lack of any significant inflammatory response, as well as the absence of excessive neointimal formation in the animals treated with nanoparticles compared with control animals. In the second phase of this study we have shown that nanoparticles can be loaded with dexamethasone and achieve a significantly high drug concentration at the delivery site with almost no systemic levels after the first 24 hours. Detectable local levels of drug persist for up to 14 days after a single short infusion. With the use of this local drug delivery system, dexamethasone significantly decreased neointimal formation after balloon injury in this experimental model of vascular injury.

Local Drug Delivery
Newer approaches for a more effective site-specific drug delivery are being directed toward local delivery systems rather than systemic drug administration in an attempt to achieve and sustain high tissue levels of the drug at the site of balloon injury and decrease or avoid the potential adverse systemic side effects. Our laboratory has recently shown that local dexamethasone, delivered with the use of a periadventitial silicone polymer system after vascular injury in the rat carotid model, markedly reduced neointimal formation compared with the adjacent injured segment without local treatment.¹⁰ Although this periadventitial approach also has been used by other investigators with successful results in the prevention of neointimal formation after vascular injury,^{11 12 23} the clinical application of such an approach in the restenosis process after coronary angioplasty is limited for obvious reasons. Endoluminal delivery is a much more practical and clinically transferable approach. Drug-releasing polymeric stents and polymer-coated stents are under investigation in different animal models¹⁶ ; however, these systems have potential disadvantages. First, most of the current stent designs cover only 5% to 12% of the arterial surface, limiting the amount of surface area in contact with the drug.²⁴ Second, and perhaps more important, is that many of the polymeric materials used for retention and continuous delivery of the agent tested in different animal models do not appear to be biocompatible with vascular tissue, as evidenced by induction of a significant inflammatory and proliferative response.^{25 26} In addition, use of a prosthetic, permanent stent implant is undesirable for the purpose of a short-term drug delivery. Balloon catheters for endoluminal local drug delivery may be more widely applied after coronary interventions. However, these systems have the disadvantages of not being capable of achieving high local tissue drug concentration, result in significant systemic drug levels, and most importantly, are unable to maintain therapeutic local drug levels for an adequate period of time.^{17 18 19 20}

Nanoparticles as Carrier
The previously mentioned limitations could be overcome if a carrier could transport the agent into the injured vessel and increase the residence time. The development of such carriers has been a major challenge. To be effective in the prevention of the restenosis process, a carrier has to meet several features. First, it is essential that the size be small enough for delivery through the current catheter designs. Second, it should have a high drug loading capacity. Third, since it cannot be removed later from the lesion, as such, it should be biodegradable. Fourth, it should gradually release the agent over an extended period of time. Fifth, it must be fully biocompatible with the vascular tissue.

The PLGA nanoparticles used in this study as carriers for continuous local drug delivery possess most of the above-mentioned characteristics. PLGA is a biodegradable polymeric material in which different agents can be incorporated. Previous investigators have shown PLGA to be biocompatible with human tissue, having been used for surgical purposes (implants, sutures) with extensive safety documentation.^{21 22 27} In the present study, the particles had a mean diameter of 165±39 nm, a significantly smaller size that can be delivered through commercially available balloon catheters (with hole sizes that range from 1 to 100 µm).¹⁶ The drug contained within the particles is gradually eluted by diffusion through pores in the particle and subsequently during the breakdown of the biodegradable material.²⁸ By changing the chemical composition and molecular weight of the polymer, the degradation time of the particle and the release kinetics of the agent can be controlled, making this system very attractive for sustained drug delivery.²⁹

In this study, we detected nanoparticles in the luminal, medial, and adventitial layers of the artery during the first 24 hours after a single 3-minute injection. Thereafter, nanoparticles appear to persist only in the adventitial layer for approximately 7 days. This layer appears to act as a reservoir for the particles. Successful delivery to the media of antiproliferative agents and oligonucleotides from the adventitia has been described previously by our group and others.^{12 30} No fluorescent activity was found outside of the adventitial layer after 24 hours. Furthermore, given the nanometer range of the particles, it is possible that the resolution power of the fluorescent microscopy technique is not sensitive enough to detect individual particles in the different layers and that only particle clusters are visible. To further evaluate the distribution and persistence of nanoparticles in the arterial wall, we have determined the presence of associated drug (dexamethasone) in the vessel wall. Despite the very low fluorescent activity at 7 days and absence of fluorescent activity at 14 days after nanoparticle infusion, a significant amount of the drug was still present in the vessel wall, suggesting that particles continue releasing the drug for at least 14 days. Whether the drug detected in tissue was due to the presence of particles in the media, intima, or adventitial layer was not clearly determined. An important finding of this study is that despite a very high level of dexamethasone in the treated segment, no systemic dexamethasone levels were detected after the first 3 hours of infusion, reflecting the true localized activity of the system.

Nanoparticles were mainly present in the lumen surface of the vessel and in the adventitial layer. Only in a limited number of segments, particles were observed in the innermost portion of the media. This discontinuous distribution of the particles suggests that the access to the adventitia was other than direct penetration through the layers of the vessel wall. These results are consistent with a recent report by Rome et al,³¹ which showed that particles between 90 and 500 nm in size delivered via a double balloon catheter accumulate in the intimal and adventitial layers with almost no particles present in the media. They have found that the mechanism by which the particles reach the adventitia was via the vasa vasorum of the artery. The present findings suggest that the adventitia function as a reservoir for the particles and that the dexamethasone diffuses from the adventitia inward toward the media. Transport of labeled albumin and heparin from the adventitia to the medial layer has been demonstrated.^{12 32}

The Problem of Polymer Biocompatibility
In a recent collaborative study, several different nondegradable and biodegradable polymeric-coated stents were deployed in pig coronary arteries to evaluate the vascular tissue response. A severe inflammatory reaction was noted with the use of each of the polymers tested including PLGA, with a massive infiltration of mononuclear cells, neutrophils, and multinuclear giant cells. No cytotoxic effect as the result of residual solvents or other chemical impurities was found.²⁵ Dev et al²⁶ recently have reported that polylactic acid microparticles (1 to 5 µm in size) were successfully delivered with the use of a microporous balloon catheter in the rabbit carotid artery and persisted in the arterial wall for at least 4 weeks after infusion. However, these particles were associated with some degree of inflammatory response and increase in intimal formation. In the present study, we have not seen a significant inflammatory response in animals treated with nanoparticles, and this biocompatibility is further supported by the lack of incremental neointimal formation. The explanation for the differences may be related to the size of the particles used in each experiment; this postulates that the inflammatory response was mainly related to a foreign body reaction rather than toxic reaction to the polymer and polymer degradation products per se. The particle size used in the present study was 10 to 100 times smaller than in the study by Dev and associates. PLGA degrades by hydrolytic cleavage of their ester linkages resulting in D-lactic/L-lactic and glycolic acid, both normal metabolic compounds.

Dexamethasone Nanoparticles and Neointimal Proliferation Inhibition
The study shows that local intravascular delivery of dexamethasone significantly decreases neointimal hyperplasia after balloon injury in the rat carotid injury model. In addition, we have demonstrated that the inhibition of the hyperplastic response was not due to a systemic effect of the drug, as evidenced by the lack of inhibited intimal growth when the nanoparticles were injected intraperitoneally.

We chose dexamethasone as an agent to test the efficacy of the nanoparticle drug delivery system on the basis of our previous experience in which periadventitial delivery of dexamethasone significantly decreased intimal growth in the same animal model.¹⁰ Although this study does not address the specific mechanism by which glucocorticoids decrease neointimal thickness, several potential explanations could be considered. First, glucocorticoids inhibit the autocrine production of platelet-derived growth factor (PDGF) A chain.^{33 34} PDGF is a potent mitogen and important stimulus for migration of smooth muscle cells.^{35 36} Second, glucocorticoids selectively inhibit the transcription of the interleukin-1ß (IL-1ß) gene and decrease the stability of IL-1ß mRNA.³⁷ IL-1ß is a cytokine that has been shown to stimulate smooth muscle cell proliferation.^{38 39} Third, glucocorticoids can suppress the activity of nuclear proteins such as c-jun/AP-1, implicated in diverse aspects of cell growth.^{40 41} Fourth, glucocorticoids decrease the inflammatory response,^{42 43} reduce extracellular matrix deposition, and may be further involved in the remodeling process by inducing the expression of metalloproteinases.^{44 45 46 47 48 49}

Conclusions
In this study we have shown that nanoparticles successfully penetrated into the vessel wall and persisted up to 14 days after a short intraluminal infusion. The PLGA nanoparticles appear to be biocompatible without engendering an intimal hyperplastic reaction. Local administration of the particles with incorporated dexamethasone was associated with a significant decrease in neointimal formation. Studies in larger animal models of restenosis using the currently available percutaneous endoluminal local delivery systems are warranted to determine the potential application of nanoparticles as an effective local delivery system after percutaneous coronary intervention in humans.

	Acknowledgments

 
This work was supported (University of Michigan) in part by a grant from Schneider, Inc, Minneapolis, Minn. The authors thank Farhad Forudi for his technical assistance in performing the animal experiments.

Received November 2, 1995; revision received March 13, 1996; accepted March 26, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 
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