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DOI: 10.1148/radiol.2282020539
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(Radiology 2003;228:555-559.)
© RSNA, 2003

Technical Developments

In Vivo US Monitoring of Catheter-based Vascular Delivery of Gene Microspheres in Pigs: Feasibility1

Xiangying Du, MD, Yuesong Yang, MD, PhD, Catherine Le Visage, PhD, Hunter H. Chen, BS, Robert DeJong, BS, Bensheng Qiu, PhD, Danming Wang, MD, Kam W. Leong, PhD, Ulrike M. Hamper, MD, MBA and Xiaoming Yang, MD, PhD

1 From the Departments of Radiology (X.D., Y.Y., R.D., B.Q., D.W., U.M.H., X.Y.) and Biomedical Engineering (C.L.V., H.H.C., K.W.L.), Johns Hopkins University School of Medicine, 720 Rutland Ave, Traylor Bldg, Rm 330, Baltimore, MD 21205. Received May 9, 2002; revision requested July 11; final revision received October 22; accepted December 10. Supported in part by a Johns Hopkins Radiology Molecular Imaging grant and National Institutes of Health grants R01 HL66187 and R01 67195. Address correspondence to X.Y. (e-mail: xyang@mri.jhu.edu).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
In this study, the authors tested the feasibility of using ultrasonography (US) to monitor catheter-based vascular gene microsphere delivery. Polymeric biodegradable microspheres (mean diameter, 5 µm) were prepared by using a double-emulsion technique to encapsulate DNA-plasmid–encoding green fluorescent protein (GFP) genes. With use of gene-delivery catheters, GFP microspheres were locally delivered into the left femoral arterial walls of six pigs; the contralateral arteries were not infused with microspheres and thus served as negative control vessels. The delivery procedures were monitored with high-frequency (8–15-MHz) transducer US. The effectiveness of monitoring with US was compared with the effectiveness of monitoring with immunohistochemical anti-GFP staining. A highly echogenic "star burst" sign around the entire vessel wall was seen at US and correlated with immunohistochemical findings that showed the destination of the gene microspheres. Study results demonstrate the potential of US for monitoring catheter-based vascular gene microsphere delivery in vivo.

© RSNA, 2003

Index terms: Animals • Genes and genetics • Microspheres • Ultrasound (US), experimental studies, 92.12981, 92.12982, 92.12983, 92.12988, 92.12989


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Gene therapy is a rapidly expanding field with great potential for the treatment of cardiovascular diseases (1). Precise delivery of gene vectors and adequate tracking of gene expression in targeted atheroscleroses are two challenges for successful vascular gene therapy. Catheter-based delivery is a promising therapeutic approach to localizing a high dose of a transgene at a targeted site while minimizing any undesirable systemic transfection. However, in vivo imaging methods of monitoring the efficacy of vascular gene vector transfer need to be developed so that one can visualize (a) where the gene vectors go, (b) how the gene vectors interact with the lesions, and (c) how long the genes function at the target.

To date, most investigations of gene therapy imaging have focused on noncardiovascular systems and primarily on neoplasms of different organs and systems (2). The reasons for this are related to the anatomic and physiologic characteristics of the cardiovascular system—specifically (a) thin vessel walls, which require high-spatial-resolution imaging modalities; (b) cardiac beating or vessel pulse, which requires specific methods to reduce motion artifacts; (c) blood flow, which requires strategies to enhance the interaction between gene vectors and the targeted vessel lesions; and (d) relatively complicated endovascular interventional procedures. Recently, the monitoring of vascular gene delivery with magnetic resonance (MR) imaging has been reported (3). The purpose of our study was to evaluate the use of ultrasonography (US) for monitoring catheter-based vascular gene vector delivery procedures in vivo.


   Materials and Methods
TOP
 ABSTRACT
 INTRODUCTION
 Materials and Methods
Results
 Discussion
 REFERENCES
 
We used 12 arteries (six examination and six control arteries) in six living domestic pigs (Archer, Belcamp, Md) that weighed approximately 16–18 kg. All animals were treated according to principles outlined by the National Society for Medical Research and the National Institutes of Health (4). In addition, the animal care and use committee at our institution approved the experimental protocol.

Study Design
This study had three parts: First, we created echogenic biodegradable microspheres that encapsulate green fluorescent protein (GFP) gene–plasmid. Second, by using a catheter-based approach, we delivered the GFP microspheres into the femoral arterial walls of the pigs and monitored the delivery with high-resolution color Doppler US (Fig 1). Third, we harvested the targeted vessels to identify the GFP transgene expression at immunohistochemical staining. The corresponding targeted vessels on the contralateral side were not infused with GFP microspheres and thus served as negative control vessels for US and immunohistochemical comparisons. In this study, we designed the microsphere to function as both a US contrast agent and a nonviral gene-carrying vector.



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  		Figure 1. Schematic diagram of the study design for the development of US-based microsphere-mediated vascular gene delivery.

 
Preparation of Echogenic Gene Microspheres
Polymeric biodegradable microspheres composed of poly(methylidene malonate 2.1.2) (Virsol, Paris, France) were created by using the double-emulsion technique (5,6): 50 mg of polymer was dissolved in 1.5 mL of ethyl acetate and sonically mixed with 150 µL of GFP reporter gene–plasmid (10 mg/mL, pEGFP-N1; Clontech, Palo Alto, Calif). For the second emulsion, the polymer, ethyl acetate, and GFP gene–plasmid mixture was added to poly(vinyl alcohol) (2% wt/vol) and stirred at room temperature for at least 14 hours to ensure complete evaporation of the ethyl acetate. The microspheres were then washed five times. The particles were finally stored as a 50 mg/mL particle-water suspension.

The size distribution and mean diameter of the particles were determined by using a Coulter counter Multisizer II (Beckman Coulter, Fullerton, Calif) fitted with a 50-µm orifice tube. The morphologic features of the particles were determined by performing scanning electron microscopy (SEM-1810; Amray, Bedford, Mass) of the freeze-dried suspension by using an electron acceleration of 20 kV. At scanning electron microscopy, the microspheres had a mean diameter of 5 µm, a spherical shape, and a smooth surface. The amount of GFP-plasmid-DNA encapsulated in the microspheres was measured with a spectrophotometer after the DNA was extracted from the microspheres. The stability of the DNA was also checked with electrophoresis. The microspheres contained 100 µg of DNA in 1 mL of the microsphere suspension (50 mg/mL).

To confirm the ultrasonic property of the GFP microspheres, we used an 18-gauge needle to inject a 2-mL suspension of the GFP microspheres (50 mg/mL) into three different sites in the right buttock of a pig. By using the same method, we also injected 2 mL of saline into three different sites of the left buttock in the same pig; these served as control sites. The entire injection procedure was monitored by using a US imaging system (model 5200; Siemens, Mountain View, Calif) with a 7.5-MHz linear-array transducer.

In Vivo Experiments
Anesthesia.—The pigs were sedated with an intramuscular injection of a mixture of 22 mg of ketamine hydrochloride (Fort Dodge Animal Health, Fort Dodge, Iowa) per kilogram of body weight, 1.1 mg/kg of acepromazine maleate (Fermenta Animal Health, Kansas City, Mo), and 0.05 mg/kg of atropine sulfate (American Regent Laboratories, Shirley, NY). An ear vein was cannulated to maintain hydration with sterile saline. Pentobarbital sodium (Abbott Laboratories, North Chicago, Ill) (20 mg/kg) was later administered intravenously to put the animal in a state of anesthesia suitable for surgery. The animals were intubated and mechanically ventilated by using an anesthesia machine that delivered 1.5%–2.0% isoflurane (Ohmeda, Liberty Corner, NJ), and their body temperature was maintained at 37°C with a water-jacket heating pad. The animals also received 100 IU/kg of heparin to achieve an activated partial thromboplastin time of longer than 80 seconds. We monitored anesthesia during the experiment by regularly performing eyelid reflex and mild paw compression tests.

Catheterization.—By using an arteriotomy approach, we cannulated a 9-F introducer into the aorta through the right carotid artery. Then, a 4-F pigtail angiographic catheter was positioned in the abdominal aorta. By injecting 20 mL of diatrizoate meglumine (60% Hypaque; Nycomed Amersham, Princeton, NJ) at a rate of 10 mL/sec, we obtained a conventional angiogram that encompassed the pelvic and femoral arteries on both sides. We then selected a 3.0–3.5-mm-diameter, 2-cm-long portion of the left femoral artery as the gene microsphere target vessel. The portion of the right femoral artery that corresponded to the targeted portion of the left femoral artery was then harvested, and it served as the negative control vessel.

Next, a 3.5–4.0-mm-diameter gene-delivery catheter (Remedy Catheter; Boston Scientific, Boston, Mass) was introduced through a 0.014-inch guide wire and positioned at the portion of the left femoral artery targeted for gene microsphere delivery. This catheter has a centrally located angioplasty balloon that is surrounded by multiple gene-delivery infusion channels that have 30-µm micropores on the surfaces (Fig 2). During catheter-based gene microsphere delivery, the inflation of the angioplasty balloon stopped blood flow in the targeted vessel portion completely; therefore, no gene microspheres entered the downstream blood circulation, lungs, or liver. The diameter ratio between the targeted arterial portion and the gene-delivery balloon was 3.0 mm/3.5 mm or 3.5 mm/4.0 mm.



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  		Figure 2. Gene microsphere delivery catheter. A, Diagram of the balloon portion of the catheter. B, Corresponding longitudinal color Doppler US image (13-MHz transmit frequency with autoadjustable 8-15-MHz linear-array transducer) of the inflated gene-delivery balloon. US probe is parallel to long axis of vessel. Structural details of the balloon, including the angioplasty balloon itself (star), the gene-infusion channels (between open arrows), and the guide wire (long arrow), can be differentiated on the US image. Infused microsphere flow (short solid arrows) at the micropores of the balloon also is seen.

 
US imaging.—We first inflated the angioplasty balloon of the catheter with saline at 4-atm support pressure. Then, a syringe pump (Harvard, Holliston, Mass) was used to infuse a 1.5–2.0-mL GFP-plasmid microsphere suspension (50 mg/mL) containing approximately 150–200 µg of GFP-plasmid into the left femoral arterial walls of the pigs at 10 mL/h for 6–8 minutes. The parameters for delivering the gene vectors to the arterial walls with the gene-delivery catheters have been previously tested and established elsewhere (3). We acquired color Doppler US images of the transfected arteries and contralateral nontransfected arteries before, during, and after microsphere infusion by using a US imaging system (Sequoia 512; Acuson/Siemens, Mountain View, Calif) with an autoadjustable 8–15-MHz linear-array transducer.

To precisely align the US images with the immunohistochemical slides, we used two kinds of markers. First, at conventional angiography, we selected a 2-cm-long nonbranched portion of the femoral artery as the vessel targeted for gene microsphere delivery. The proximal end of the targeted arterial portion had a large branch. Thus, by using the location of this branch, we could easily localize the targeted arterial portion when it was exposed surgically. Second, we made two surface markers with sutures on the skin. The sutured spots were marked along the targeted arterial portions with US guidance and identified as crossing the central portion of the balloon. The distance between the two markers was the same as the 1-cm thickness of the US transducer.

Immunohistochemical Confirmation
After the GFP-plasmid microspheres were infused, the pigs were kept alive for 5 days to allow sufficient GFP gene expression. Previous study (7) results confirmed that GFP genes achieve peak expression in the vasculature 4–5 days after primary gene delivery. On the basis of this information, we chose postdelivery day 6 to harvest the targeted arterial tissues for immunohistochemical analysis. On day 6, the GFP-transfected arterial portions were harvested according to the branch and suture markers. The pigs were then euthanized with intravenous injection of pentobarbital (100 mg/kg). The 1-cm-long middle portion of the harvested arteries was sliced for immunohistochemical staining. GFP-transfected and nontransfected vessel tissues were immediately frozen in liquid nitrogen and then sectioned for placement onto 14-µm slides. The slides were fixed in 100% ethanol for 20 minutes. After air drying, the slides were treated with 3% H2O2 in phosphate-buffered saline (pH 7.4) for 20 minutes to quench endogenous peroxidase activity.

After nonspecific sites were blocked with 10% goat serum in phosphate-buffered saline for 60 minutes, the slides were washed with phosphate-buffered saline three times for 5 minutes and incubated overnight with the specific monoclonal antibody for GFP (dilution 1:250; Roche, Indianapolis, Ind) at 4°C. After washing off the unbound primary antibodies with phosphate-buffered saline, we incubated the slides with biotinylated antimouse antibody (1:500 dilution) for 1 hour. Specific binding was detected by using a 1:100 dilution of avidin-biotin complex (Vector Laboratories, Burlingame, Calif), a substrate H2O2 solution, and a diaminobenzidine kit for 1 hour, according to manufacturer instructions. The specimens were then counterstained with hematoxylin, dehydrated with gradient alcohol and xylenes, fixed under coverslips, and examined under a microscope (VANOX AHBS3; Olympus, Dulles, Va) (7).

The color Doppler US image and immunohistochemical findings in the nontransfected and transfected tissues were read independently by two investigators (X.D., X.Y.). Contrast material enhancement and immunohistochemical staining were evaluated by using a qualitative grading system: 0 meant no echo signal enhancement or GFP staining identifiable; 1, weak or only partial echo signal enhancement or GFP staining identifiable; and 2, good or sufficient echo signal enhancement or GFP staining around the entire gene microsphere–targeted vessel wall. The resulting grades for the US and immunohistochemical findings were then integrated.


   Results
TOP
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Intramuscular saline injection produced no echogenic signals at any of the three sites in the pig, whereas microsphere injection produced a highly echogenic area in the targeted arterial portion at all three sites. This area confirmed the echogenic property of the microspheres (Fig 3). At color Doppler US, we were able to visualize the entire process of balloon positioning, inflation, and deflation in real time. These US images, which were obtained by using the 8–15-MHz transducer, enabled us to differentiate the structural details of the balloon, including the angioplasty balloon itself, the gene-delivery-infusion channels and their micropores, and the guide wire (Fig 2).



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  		Figure 3. Color Doppler US images obtained with a 7.5-MHz linear-array transducer before (A) and after (B) after intramuscular injection of microspheres into a pig. US probe is parallel to long axis of needle. The injected microspheres are depicted as a local hyperechogenic area (arrow in B). The arrow in A points to the injection needle.

 
In all six GFP microsphere–transfected vessels, we visualized dynamically the echo signal increase around the entire targeted vessel wall, which started immediately after the gene microsphere infusion. On the transverse color Doppler US images obtained during infusion, the microspheres delivered to the targeted vessel wall had a highly echogenic "star burst" appearance around the entire vessel wall (Fig 4, A). Color Doppler US enabled us to visualize the motion echo signals at the micropores of the gene infusion channels; these signals were caused by the flow of microspheres from the gene infusion channels to the targeted vessel wall (Fig 2, B). Postinfusion US images showed the echogenic enhancement of the targeted vessel walls that was caused by the microspheres (Fig 5). In one case, color Doppler US depicted perforation in the target wall, which may have been an indication of balloon dilation pressure–driven delivery-induced damage (Fig 5).



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  		Figure 4. Comparison of US and immunohistochemical monitoring of vascular gene microsphere delivery. A, Color Doppler US image of femoral artery obtained with autoadjustable 8-15-MHz linear-array transducer shows GFP microspheres flowing through the micropores of the balloon and appearing as a highly echogenic star burst ring (arrow) that is evenly distributed around the entire vessel wall. US probe is perpendicular to vessel axis. B, Corresponding GFP expression—that is, the brown precipitate surrounding the entire targeted vessel wall (between arrows)—in the immunohistochemically stained specimen confirms the US findings. (Original magnification, x10.)

 


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  		Figure 5. High-frequency-transducer (13-MHz transmit frequency with autoadjustable 8-15-MHz linear-array transducer) color Doppler US images obtained before (A, B) and after (C, D) catheter-based intravascular infusion of gene microspheres into the femoral artery of a pig. In A and C, US probe is parallel to long axis of vessels. In B and D, US probe is perpendicular to long axis of vessels. A, B, Guide wire (arrows in A) is placed in the lumen of the femoral artery. C, D, Targeted arterial wall (open arrow in C) is echogenically enhanced. In C, the region in the box highlights a perforation (solid arrow) in the posterior wall that was not observed on the preinfusion images. A (in B and D) = femoral artery, V = femoral vein.

 
Immunohistochemical analysis of the tissues transfected with GFP-plasmid microspheres revealed local GFP transgene expression in all six vessels. This expression appeared as brown precipitates in the endothelial cells and smooth muscle cells of the intima and media layers of the targeted vessel wall. These brown GFP precipitates were not detected in the six nontransfected tissues (Fig 6). In all six cases, GFP microsphere–induced enhancement of the targeted vessel walls at color Doppler US (grade 2 for all six targeted vessels) correlated with GFP staining of the targeted vessel tissues at immunohistochemical analysis (grade 2 for all six targeted vessels) (Fig 4).



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  		Figure 6. Immunohistochemically stained specimens of nontransfected (A) and GFP-transfected (B) arteries. GFP is seen as brown precipitates in the endothelial cells and smooth muscle cells from the intima to the media. (Original magnification, x200.)

 

   Discussion
TOP
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results
 Discussion
REFERENCES
 
US has prominent advantages over other imaging modalities, including relative technical simplicity, portability, cost-effectiveness, real-time imaging, and lack of radiation. In addition, results of several experimental studies have demonstrated that US with ultrasonic contrast agents or tracers, such as echogenic lipid-based liposomes and air-containing microbubbles, can improve the sensitivity of diagnostic imaging of specific targets, such as fibrin in thrombi (8), and/or improve gene expression by adapting US energies (9). Before the present study, the use of US to monitor catheter-based vascular gene vector delivery in vivo had not been explored. However, US also has pitfalls, including operator dependence, required technical expertise, and difficulty in quantitatively estimating signal intensity. In our study, the color Doppler US images were acquired by an experienced radiologist to maintain consistent image quality, and the acquired images were interpreted by two other investigators to prevent possible bias in the readings.

Clinical imaging of vascular gene therapy includes two components: (a) assessing the success of the primary gene vector delivery procedure and (b) tracking the vascular gene expression several days after the primary gene vector delivery. Our current study was focused only on the first component—that of monitoring the primary gene microsphere delivery procedure. Monitoring the delivery of the vectors is critical to the success of vascular gene therapy. After catheter-based delivery, clinicians need to immediately evaluate the success of the procedure. This evaluation includes (a) confirming where the gene vectors have been delivered, (b) determining how satisfactorily the targeted portion of the vessel wall has been marked, and (c) determining whether the delivery procedure has caused complications, such as the perforations illustrated in Figure 5. Failure to recognize unsuccessful delivery can delay effective treatment for several months, whereas if a deficiency in the procedure is detected, it can be remedied immediately with use of alternative treatments.

Our study results represent encouraging initial evidence that catheter-based vascular gene microsphere delivery can be dynamically monitored in vivo with US. In our technical development study, the microspheres were used as both US contrast agents and gene-carrying vectors. Thus, US findings of uneven microsphere distribution should indicate insufficient gene localization in a primary gene microsphere delivery procedure that was unsuccessful because of problems related to either the gene-delivery device or a component of the diseased vessel wall itself, such as a calcified atherosclerotic plaque that prevents genetic materials from entering the "hard" vessel portion.

The color Doppler US images obtained with the 8–15-MHz transducer enabled us to visualize the entire process of gene-delivery balloon positioning, inflation, and deflation. With US, both the infusion of the microspheres through the balloon micropores and the delivery-induced perforations in the vessel wall could be seen. The expression of GFP observed in situ with immunohistochemistry indicated functional in vivo gene transfer and expression with our microsphere-mediated vascular gene transfection approach. These findings may be based on the fact that the microspheres not only reflected sound waves, but they also absorbed sonic energy, and this capability helps to enhance gene transfection and expression. Several authors have observed the possible mechanisms behind the US enhancement of gene transfection, including (a) ultrasonic heating of and ultrasonic shock waves to the cell membrane that facilitate passage of the microparticles across the membranes (10), (b) ultrasonic cavitations that increase the gene-drug release from the microparticles (11), and (c) ultrasonic effects on cell regulation or transcription factors (12). However, further study is required to delineate the exact mechanisms of US enhancement of gene transfection.

In conclusion, we believe that US has potential as an imaging tool to monitor catheter-based vascular gene microsphere delivery in vivo.


   ACKNOWLEDGMENTS
 
The authors thank Mary McAllister for her editorial assistance. The support of Virsol for providing the polymer is gratefully acknowledged.


   FOOTNOTES
 
Abbreviation: GFP = green fluorescent protein

Author contributions: Guarantors of integrity of entire study, X.Y., K.W.L., U.M.H.; study concepts and design, all authors; literature research, X.D., Y.Y., C.L.V.; experimental studies, all authors; data acquisition, X.D., Y.Y., B.Q., X.Y.; data analysis/interpretation, X.D., Y.Y., R.D., U.M.H., X.Y.; manuscript preparation and definition of intellectual content, all authors; manuscript editing, X.D., X.Y., C.L.V.; manuscript revision/review and final version approval, all authors


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

  1. Nabel E, Leiden J. Gene transfer approaches for cardiovascular disease. In: Chien K, eds. Molecular basis of cardiovascular disease. Philadelphia, Pa: Saunders, 1999; 86-112.
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  4. National Institutes of Health Publication no. Guide for the care and use of laboratory animals 80-23. Bethesda, Md: National Institutes of Health, 1985.
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  7. Yang X, Liu H, Li D, et al. Digital optical imaging of green fluorescent proteins for tracking vascular gene expression: feasibility study in rabbit and human cell models. Radiology 2001; 219:171-175.[Abstract/Free Full Text]
  8. Lanza G, Wallace K, Scott M, et al. A novel site-targeted ultrasonic contrast agent with broad biomedical application. Circulation 1996; 94:3334-3340.[Abstract/Free Full Text]
  9. Lawrie A, Brisken A, Francis S, et al. Ultrasound enhances reporter gene expression after transfection of vascular cells in vitro. Circulation 1999; 99:2617-2620.[Abstract/Free Full Text]
  10. Greenleaf W, Bolander M, Sarkar G, Goldring M, Greenleaf J. Artificial cavitation nuclei significantly enhance acoustically induced cell transfection. Ultrasound Med Biol 1998; 24:587-595.[CrossRef][Medline]
  11. Shohet R, Chen S, Zhou Y, et al. Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium. Circulation 2000; 101:2554- 2556.[Abstract/Free Full Text]
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