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Influence of Needle Gauge On In Vivo Ultrasound and Microbubble-Mediated Gene Transfection
Ultrasound in Med. & Biol., Vol. 37, No. 9, pp. 1531–1537, 2011 Copyright Ó 2011 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/$ - see front matter
doi:10.1016/j.ultrasmedbio.2011.05.019
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Original Contribution INFLUENCE OF NEEDLE GAUGE ON IN VIVO ULTRASOUND AND MICROBUBBLE-MEDIATED GENE TRANSFECTION RICHARD J. BROWNING,* HELEN MULVANA,* MENGXING TANG,y JO V. HAJNAL,* DOMINIC J. WELLS,z and ROBERT J. ECKERSLEY* * Imaging Sciences Department; y Department of Bioengineering, Imperial College London; and z Department of Veterinary Basic Sciences, Royal Veterinary College, London, UK (Received 1 November 2010; revised 11 May 2011; in final form 16 May 2011)
Abstract—Ultrasound and microbubble-mediated gene transfection are potential tools for safe, site-selective gene therapy. However, preclinical trials have demonstrated a low transfection efficiency that has hindered the progression of the technique to clinical application. In this paper it is shown that simple changes to the method of intravenous injection can lead to an increase in transfection efficiency when using 6-MHz diagnostic ultrasound and the ultrasound contrast agent, SonoVue. By using needles of progressively smaller gauge, i.e., larger internal diameter (ID), from 29 G (ID 0.184 mm) to 25 G (ID 0.31 mm), the transfection of a luciferase plasmid (pGL4.13) was significantly increased threefold in heart-targeted female CD1 mice. In vitro work indicated that the concentration and size distribution of SonoVue were affected by increasing needle gauge. These results suggest that the process of systemic delivery alters the bubble population and adversely affects transfection. This is exacerbated by using high-gauge needles. These findings demonstrate that the needle with the largest possible ID should be used for systemic delivery of microbubbles and genetic material. (E-mail: [email protected]) Ó 2011 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound, Gene transfection, Microbubble, Needle gauge, Luciferase, In vivo bioluminescence, Systemic delivery.
It has long been established that high-intensity ultrasound can be used to induce transient pores in cells, a process known as sonoporation (Apfel and Holland 1991; Guzman et al. 2001). This can lead to uptake of any codelivered drug or genetic products (Kim et al. 1996; Sakakima et al. 2005; Schlicher et al. 2006; van Wamel et al. 2006). Later studies demonstrated that the concurrent use of ultrasound contrast agents (Greenleaf et al. 1997; Li et al. 2003) lowered the intensities required to generate the effect to within the recommended safe levels of exposure currently used in clinical ultrasound (Haar 2002; Duck 2008). Ultrasound contrast agents are shell encapsulated gas microbubbles, typically 1–10 mm in diameter, which oscillate when exposed to diagnostic ultrasound. Such oscillations generate bioeffects in vivo, which allow the transfer of genetic material or drugs through cell membranes and across blood vessel walls. With the established safety of ultrasound (Duck 2008) and the ability to target transfection to specific sites in the body, ultrasound and microbubble-mediated gene transfection (UMGT) has generated a lot of interest. Many preclinical studies
INTRODUCTION Viral vectors have commonly been used in preclinical and clinical gene therapy trials to investigate the possibility of treating a number of acquired or hereditary disorders (Culver et al. 1991; Deodata et al. 2002; Galeano et al. 2003; Bainbridge et al. 2008). However, the potentially severe immunological and oncogenic side effects associated with viral vectors (Marshall 1999; Hacein-Bey-Abina et al. 2003; Marshall 2003) have led to the development of safer nonviral vectors, such as naked DNA. These are well-tolerated by host organisms (Wolff et al. 1990) but not readily taken up by cells. Thus, other physical or chemical techniques have been developed to increase the transfection efficiency without the use of viral vectors, while retaining the biosafety advantage (Glover et al. 2005; Labas et al. 2010).
Address correspondence to: Mr Richard J Browning, Room 214B ISD Labs, 2nd Floor Francis Fraser Building, Hammersmith Hospital, Hammersmith Campus, Imperial College London, Du Cane Road, London, W12 0NN, UK. E-mail: [email protected] 1531
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have investigated its potential to treat a variety of disorders and diseases (Chen et al. 2003; Alete 2008; Alter et al. 2009; Chai et al. 2009; Li et al. 2009; Wang et al. 2009). However, the technique has relatively low transfection efficiencies, especially in compared with other nonviral gene therapy techniques (Wells 2004; Kusumanto et al. 2007), which has hindered its development toward clinical application. In vivo administration of microbubbles is generally performed by injection into the blood supply either as a bolus or infusion. It has been shown in vitro that lipid-shelled microbubbles are susceptible to changes in pressure and shear stresses that occur during passage through syringes and needles (Barrack and Stride 2009). The concentration and size distribution of microbubble suspensions can be significantly decreased and altered, particularly when using large-gauge (small-bore) needles around 27–30 G (Talu et al. 2008). Few preclinical papers state the needle size used; however, because the preclinical model is typically a rodent, the needle size will usually be around 27–29 G (Sakakima et al. 2005; Wang et al. 2005). Considering the in vitro influence on microbubble size and concentration, there could be significant implications using such needles for UMGT. This may also contribute to the low transfection efficiencies often reported in vivo (Alete 2008; Wang et al. 2009; Phillips et al. 2010). This paper investigates the influence of needle gauge on transfection in vivo and presents simple methodological changes to improve the technique. MATERIALS AND METHODS Ultrasound contrast agent SonoVue (Bracco Research SA, Geneva, Switzerland) is a lipid-shelled microbubble of sulphur hexafluoride gas supplied as a lyophilized powder with 5 mL of saline for reconstitution. When prepared according to the manufacturer’s specification, the microbubble concentration ranges from 2–5 3 108 microbubbles/mL, with a mean diameter of 2.5 mm (Schneider 1999). In all experiments, SonoVue at double the normal concentration was used by reconstituting the lyophilized powder with half of the supplied saline because previous studies have shown that increasing the SonoVue concentration leads to
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increased transfection. However, increasing beyond double concentration leads to increased mortality in mice (Alete 2008). Plasmid The plasmid pGL4.13 (Promega UK Ltd., Southampton, UK) was used in all in vivo experiments. It carries the Phontinus pyraelis luciferase gene, luc2, controlled by the SV40 promoter and is 4.6 kb in size. It was transformed into a DH5a Escherichia coli strain following the manufacturer’s instructions (MAX Efficiency DH5a Competent Cells, Invitrogen Ltd., Paisley, UK). Plasmid was prepared from 2.4 L, overnight cultures using Qiagen’s EndoFree Gigaprep kit (Qiagen GmbH, Hilden, Germany) following the manufacturer’s instructions. Recovered plasmid was adjusted to a concentration of 4 mg/mL in injectable water for in vivo use. Syringes and needles Three gauges of needle were tested: a 25-G needle integrated in a 1-mL syringe (SurSaver syringe, Terumo Europe N.V., Leuven, Belgium, Cat. No.: SS01D2516); a 27-G needle integrated in a 0.5-mL syringe (Tuberculin syringe, BD Medical, NJ, USA, Cat. No.: 305620); and a 29-G needle integrated in a 0.5-mL syringe (Insulin syringe, Terumo UK Ltd., Surrey, UK, Cat. No.: BS 5 05M2913) (Table 1 has the full details). Integrated needles were used to minimize dead space, which is considerable when using standard hubmounted needles. This is an important consideration during microbubble administration because microbubbles float. This buoyancy is dependent on their gas volume, and therefore size, which can result in a heterogeneous size distribution in the syringe, developing over time. A portion of the dose is left in the dead space after administration, which leads to the loss of a size section of microbubble population. By using integrated needles, the dead space otherwise introduced by the needle-syringe connection is significantly reduced. In vitro UCA characterization In vitro samples were prepared to replicate the method used for in vivo delivery; 200 mL of a freshly prepared SonoVue suspension were removed to a 1.5-mL microcentrifuge tube using a 19-G needle and 1-mL
Table 1. Properties of the three syringes in use Syringe Terumo insulin syringe BD tuberculin syringe Terumo SurSaver syringe
Needle gauge 29 G 27 G 25 G
Needle ID (mm)
Needle OD (mm)
Needle length (mm)
Syringe volume (mL)
Syringe ID (mm)
Syringe OD (mm)
0.184 0.210 0.31
0.337 0.413 0.514
13.0 12.7 16.0
0.5 0.5 1.0
3.45 3.55 4.7
5.40 5.70 6.6
ID 5 Internal diameter; OD 5 outer diameter.
Influence of needle gauge on in vivo ultrasound d R. J. BROWNING et al.
syringe. One-hundred fifty mL of the suspension was added to 50 mL of injectable water by pipette and mixed gently by stirring the pipette tip. The 200-mL sample was drawn up using the experimental syringe and held vertically for 20 s as air bubbles were removed. The syringe was held horizontally for a further 20 s to simulate the placing of the needle, which would occur in vivo, and contents expelled into an empty microcentrifuge tube over 10 s. A ‘‘no-needle’’ control sample was also prepared by pipette from the diluted microbubble suspension. Samples were then diluted fivefold in water and placed on a hemocytometer for microscopy examination. After allowing 5 min for bubbles to rise to the cover-slip, 20 images were taken using a light microscope with digital acquisition (40x magnification, Nikon Eclipse 50i, Nikon Digital Camera DXM1200C, Nikon, Tokyo, Japan). Three sample repeats were prepared per syringe or control, with each sample imaged twice. Repeat samples were interleaved over time, to monitor the effect of bubble decay within the vial on suspension characteristics. Images were analyzed in MATLAB (The MathWorks Inc., Natick, MA, USA) using a custom sizing and counting program described in Sennoga et al. (2010) to determine the concentration, mean diameter, median diameter and gas concentration. Briefly, the program segments the bubbles from the image background. Individual bubbles are sized and counted. Clustered bubbles, identified through comparison with a circle, are excluded from the distribution estimation but included in the total bubble count. For further detail, please refer to Sennoga et al. (2010). The bubble decay, observed to be approximately linear over time, was calculated from control values and used to adjust our experimental results. In vivo transfection All animal work was conducted under the authority of a UK Home office project licence as required by the Animals (Scientific Procedures) Act 1986. Mice were maintained in a minimal-disease facility fully compliant with Home Office guidelines with food and water ad libitum. Six- to 8-week-old female CD1 mice were anesthetized using 5% isoflurane gas and maintained at 2–3% isoflurane on a heat mat. Hair was removed from the chest of each mouse by shaving and application of depilatory cream, to ensure good coupling with warmed ultrasound gel. A Siemens Acuson Sequoia (Siemens plc, Surrey, UK) ultrasound scanner and 15L8 linear array transducer were used throughout. Organ location and imaging was conducted at low intensity (14 MHz, 0.06 MI, focal depth 0.75 mm). The transducer was positioned using a cranialdorsal long-axis orientation through the left side of the chest so that the acoustic focus was coincident with the heart and acoustic emission frozen to prevent premature destruction of microbubbles. As described in the
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in vitro work, a 200-mL sample of microbubbles was removed from the SonoVue vial to a microcentrifuge tube using a 19-G needle hub mounted to a 1-mL syringe. A suspension of SonoVue (150 mL) and plasmid (50 mL) was prepared in a separate microcentrifuge tube and drawn into the experimental syringe. The syringe was held vertical for approximately 20 s as air bubbles were removed. Intravenous tail vein placement took an approximate further 20 s, during which time the syringe was held horizontally. The suspension was administered by hand at an approximate flow rate of 0.02–0.04 mL/s, i.e., each 200-mL injection took between 5 and 10 s. This was immediately followed by 2 min of high-intensity ultrasound (6 MHz, 1.6 MI, focal depth 0.75 mm) exposure, with the focus swept through the entire heart by slow movement of the transducer. After treatment, the transducer was removed and the mouse was recovered in a hot box. Bioluminescence imaging Three days after transfection, the transfected mice were imaged in vivo using an IVIS 100 bioluminescence camera and Living Image V3.2 software (Caliper Life Sciences, Hopkinton, MA, USA). Mice were anesthetized using 5% isoflurane and maintained at 1–2% isoflurane on the heated imaging platform of the IVIS 100 machine. An intraperitoneal injection of the substrate luciferin (Gold Biotechnology Inc., St. Louis, MO, USA) (300 mg/kg body weight in injectable water) was administered and the resulting bioluminescence in transfected tissues expressing luciferase was imaged. Bioluminescence images were then taken every two minutes (1-min exposure, field of view 10 cm, automatic height focusing) for 20 min. Using the analysis tools of the Living Image software, a region of interest was drawn around the area of bioluminescence and the photon flux (photon/s) measured. Signals were normalized for noise by removal of the background signal measured from an untreated site on each image. Statistics The in vivo data were determined to be normally distributed by the D’Agostino-Pearson K2 test and homoscedastic by Bartlett’s test. A one-way analysis of variance (ANOVA) was performed on the different needle groups to discern whether a statistical difference existed between the groups. Pairwise comparison was performed using Tukey’s test to determine which sample groups were significantly different. Significance was assumed if p # 0.05. Statistical analysis of in vitro data was limited to those bubbles acoustically active at clinical diagnostic frequencies (dia. 0.5 to 10 mm) (Gorce et al. 2000). In vitro needle and control data were compared using the derived
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population statistics of concentration, mean diameter, median diameter and gas concentration from each of the 6 repeats. The concentration, mean diameter and gas concentration were determined to be normally distributed and parametric statistical methods were used. Nonparametric methods were used to assess median diameters. All derived population statistics were found to be homoscedastic using Bartlett’s test or the BrownForsythe test. One way ANOVAs were used to determine whether a statistical difference existed between the needle groups in terms of the concentration, mean diameter and gas concentration. A Kruskal-Wallis ANOVA was used for median diameter. The parametric Tukey’s test or nonparametric Dunn’s test was used for pairwise comparison. Significance was assumed if p # 0.05. RESULTS Effect of needle gauge on transfection in vivo The in vivo peak bioluminescence measured three days after transfection was averaged over nine mice per needle group (see Fig. 1 for example images). A trend of increasing transfection with decreasing needle gauge is apparent (Fig. 2); using 27-G needles (7.21 3 105 photon/s) causes double the transfection compared with using 29-G needles (3.57 3 105 photon/s) while using 25-G needles (1.05 3 106 photon/s) significantly increases transfection almost threefold (p , 0.01). In addition, as the needle gauge increases so does the variability, with the standard deviation increasing between 29-G needles and 25-G needles; from 2.95 3 105 photon/s to 5.23 3 105 photon/s respectively. Effect of needle gauge on microbubbles in vitro To study the effect of needle gauge on microbubble suspensions directly, microbubbles were optically examined before and after passage through a needle (Fig. 3).
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Optical examination and comparative statistics revealed that, compared with the control, a 25-G needle caused no change in bubble concentration (Fig. 3a), mean diameter (Fig. 3b), median diameter (Fig. 3c) or gas concentration (Fig. 3d); a 27-G needle caused a slight (6%) drop in concentration and gas concentration but no change in the mean or median; a 29-G needle caused a significant drop in both concentration (30%) and gas concentration (45%), but only a slight decrease in mean diameter (9.8%) and median diameter (5.9%). In addition, 25-G and 27-G needles caused no change in the size distribution, whereas 29-G needles showed a downward shift in size distribution, because of the decrease in concentration.
DISCUSSION Our in vivo results demonstrate that using smaller needle sizes reduces the maximum transfection efficiency possible for a particular dose, a finding not reported before in the literature. This was supported by our in vitro work, which demonstrated a decrease in concentration as needle size is reduced, suggesting that a change in concentration is responsible for decreased transfection. The difference in concentration after needle passage between 29 G and 25 G supports the finding of a significant difference in transfection efficiency. However, the drop in concentration through a 27-G needle is more modest compared with the change in transfection. It is possible that additional factors may contribute to a lesser extent the differences observed in vivo, such as back pressure from the blood, or changes in blood flow caused by needle intrusion. In addition, it may be that needle passage affects the long term stability of a microbubble, a factor not investigated as part of this work. Work is currently underway to model and investigate these effects in vitro.
Fig. 1. Example in vivo bioluminescence images of mice transfected using (a) a 25-G needle, (b) a 27-G needle or (c) a 29-G needle. (Scale in photons per second per cm2 per steradian.).
Influence of needle gauge on in vivo ultrasound d R. J. BROWNING et al.
Fig. 2. In vivo peak bioluminescence levels three days after MBUS treatment averaged over nine mice per sample group. (Error lines are standard deviation, *p , 0.01 vs. mice treated with 29-G needles.).
Microbubble characteristics, such as size and concentration, play an important role in transfection. The size of a microbubble determines its resonant frequency and thus how it will respond to the acoustic driving frequency used during ultrasound exposure (Miller 1998; Chomas et al. 2001), which in turn affects the transfection efficiency (Chen et al. 2003). Several studies have demonstrated the link between microbubble suspension concentration
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and transfection (Koike et al. 2005; Alter et al. 2009). Because the bioeffects of cavitating microbubbles are thought to cause transfection, an increased number of microbubbles will cause increased bioeffects. This may also be related to a greater number of microbubbles being in contact with the cell, potentially leading to increased transfection. In addition, microbubbles in vitro tend to be more stable at higher concentrations (Feshitan et al. 2009), which may allow greater in vivo persistence and may increase the likelihood of sonoporation. There is a noticeable variability in each needle group tested. This is likely is related to limitations in the nature of the transfection and subsequent acquisition of the bioluminescence. Although the ultrasound exposure was focused to deliver maximum acoustic energy at a particular depth, propagation through the over- and underlying tissues is also likely to cause some unintentional plasmid transfection. Because the animals are biologically variable, e.g., in size and weight, some variability during transducer placement is unavoidable for the focus to be coincident with the heart. This could have led to a random distribution of transfection outside of the focus. Because the SV40 promoter on the plasmid is not tissue-specific, successful transfection in any tissue will lead to expression, which is detected by the
Fig. 3. Optically measured characteristics of microbubbles after needle passage. The plots show the mean value from six repeats per needle gauge adjusted for bubble decay measuring (a) concentration (microbubbles/mL), (b) mean diameter (mm), (c) median diameter (mm) and (d) gas concentration (mL/mL). Error bars show standard deviation per experimental syringe (*p , 0.05 vs. 29-G needle, yp , 0.05 vs. 25-G needle).
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bioluminescent camera. Indeed, ex vivo analysis of dissected thoracic tissues from transfected mice did in some cases reveal low levels of luminescence in the lungs and intercostal muscles. The range of injection time durations, from 5–10 s, could also have influenced the microbubble suspension finally delivered. Although our methodology aimed to replicate injection conditions for each mouse treated, even a small variation in administration time, in conjunction with bubble buoyancy and the shear stresses exerted on these bubbles close to the syringe wall during administration, could influence the suspension characteristics. These factors could therefore contribute to the variability seen for each group. However, this does not change the observation that an increased needle diameter has caused an increase in transfection. The in vitro investigation sought to identify potential reasons for the differences in transfection efficiency observed in vivo. From previous work conducted by Talu et al. (2008), we did not expect the flow rates and initial concentrations used would influence the microbubble populations before or after needle passage. We hypothesise that the syringe position and time taken to eliminate air bubbles (needle vertical for 20 s) and position of the needle for intravenous administration (needle horizontal for 20 s) alters the homogeneity of the suspension before delivery, and that this may influence both the number of bubbles affected by the shear stresses exerted at the syringe wall and the concentration of the suspension delivered. However, because the change in bubble population only occurs for the higher-gauge needles, the needle must be the primary agent in bubble destruction. It is possible that the shear stresses imposed on the bubbles as the plunger is depressed cause bubble instability. This instability leads to destruction during needle passage, but only if the needle is of sufficiently small diameter. It is likely that needle diameter is important because of the increase in pressure gradient from syringe barrel to needle or the greater shear stress occurring within the needle. It may be expected that larger bubbles rather than smaller would be more readily destroyed (Borden and Longo 2002; Kwan and Borden 2010); however, as previously noted, we limited our statistical analysis to clinically acoustic bubbles of 1–10 mm, and over this size range needle gauge did not cause a statistically significant alteration in microbubble size distribution or median diameter. It should be noted that there are important differences between our investigation and those conducted by Barrack and Stride (2009) and Talu et al. (2008). In the Barrack and Stride study, the needles used were detachable 18 G or 25 G attached to a 5-mL or 10-mL syringe depending on the experiment. The Talu et al. study instead used catheters of six-inch polyethylene tubing
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attached to needle gauges of 23 G, 27 G and 30 G. The diameter of the tubing matched the needle gauge in use and the syringe size was unspecified. It was therefore important to perform our own in vitro experiments to effectively examine the effect our syringes with integrated needles had on microbubble suspensions. Most importantly, our findings indicate that the largest needle size permitted by the preclinical model should be used when administering ultrasound contrast agent via intravenous injection. This will cause the least change to the microbubble population and their acoustic response and subsequent bioeffects. However, it must be noted that the vein diameter itself will limit the size of needle that can be used. In mice 6–8 weeks of age, a 25-G needle was the largest needle size that we judged could be safely used for intravenous injections. For larger animal models, this limit should be less significant. CONCLUSION We have shown here that plasmid transfection using ultrasound and the contrast agent SonoVue can be improved when using needles of a larger bore. UMGT has many advantages over other commonly used transfection techniques but is hampered by low transfection efficiency. Simple changes in the methodological procedures can help improve this by increasing the sensitivity of the system, which will allow observation of other optimization effects to be observed more clearly. Acknowledgments—This work was supported by an Engineering and Physical Sciences Research Council grant (EP/F066740/1).
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Original Contribution INFLUENCE OF NEEDLE GAUGE ON IN VIVO ULTRASOUND AND MICROBUBBLE-MEDIATED GENE TRANSFECTION RICHARD J. BROWNING,* HELEN MULVANA,* MENGXING TANG,y JO V. HAJNAL,* DOMINIC J. WELLS,z and ROBERT J. ECKERSLEY* * Imaging Sciences Department; y Department of Bioengineering, Imperial College London; and z Department of Veterinary Basic Sciences, Royal Veterinary College, London, UK (Received 1 November 2010; revised 11 May 2011; in final form 16 May 2011)
Abstract—Ultrasound and microbubble-mediated gene transfection are potential tools for safe, site-selective gene therapy. However, preclinical trials have demonstrated a low transfection efficiency that has hindered the progression of the technique to clinical application. In this paper it is shown that simple changes to the method of intravenous injection can lead to an increase in transfection efficiency when using 6-MHz diagnostic ultrasound and the ultrasound contrast agent, SonoVue. By using needles of progressively smaller gauge, i.e., larger internal diameter (ID), from 29 G (ID 0.184 mm) to 25 G (ID 0.31 mm), the transfection of a luciferase plasmid (pGL4.13) was significantly increased threefold in heart-targeted female CD1 mice. In vitro work indicated that the concentration and size distribution of SonoVue were affected by increasing needle gauge. These results suggest that the process of systemic delivery alters the bubble population and adversely affects transfection. This is exacerbated by using high-gauge needles. These findings demonstrate that the needle with the largest possible ID should be used for systemic delivery of microbubbles and genetic material. (E-mail: [email protected]) Ó 2011 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound, Gene transfection, Microbubble, Needle gauge, Luciferase, In vivo bioluminescence, Systemic delivery.
It has long been established that high-intensity ultrasound can be used to induce transient pores in cells, a process known as sonoporation (Apfel and Holland 1991; Guzman et al. 2001). This can lead to uptake of any codelivered drug or genetic products (Kim et al. 1996; Sakakima et al. 2005; Schlicher et al. 2006; van Wamel et al. 2006). Later studies demonstrated that the concurrent use of ultrasound contrast agents (Greenleaf et al. 1997; Li et al. 2003) lowered the intensities required to generate the effect to within the recommended safe levels of exposure currently used in clinical ultrasound (Haar 2002; Duck 2008). Ultrasound contrast agents are shell encapsulated gas microbubbles, typically 1–10 mm in diameter, which oscillate when exposed to diagnostic ultrasound. Such oscillations generate bioeffects in vivo, which allow the transfer of genetic material or drugs through cell membranes and across blood vessel walls. With the established safety of ultrasound (Duck 2008) and the ability to target transfection to specific sites in the body, ultrasound and microbubble-mediated gene transfection (UMGT) has generated a lot of interest. Many preclinical studies
INTRODUCTION Viral vectors have commonly been used in preclinical and clinical gene therapy trials to investigate the possibility of treating a number of acquired or hereditary disorders (Culver et al. 1991; Deodata et al. 2002; Galeano et al. 2003; Bainbridge et al. 2008). However, the potentially severe immunological and oncogenic side effects associated with viral vectors (Marshall 1999; Hacein-Bey-Abina et al. 2003; Marshall 2003) have led to the development of safer nonviral vectors, such as naked DNA. These are well-tolerated by host organisms (Wolff et al. 1990) but not readily taken up by cells. Thus, other physical or chemical techniques have been developed to increase the transfection efficiency without the use of viral vectors, while retaining the biosafety advantage (Glover et al. 2005; Labas et al. 2010).
Address correspondence to: Mr Richard J Browning, Room 214B ISD Labs, 2nd Floor Francis Fraser Building, Hammersmith Hospital, Hammersmith Campus, Imperial College London, Du Cane Road, London, W12 0NN, UK. E-mail: [email protected] 1531
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have investigated its potential to treat a variety of disorders and diseases (Chen et al. 2003; Alete 2008; Alter et al. 2009; Chai et al. 2009; Li et al. 2009; Wang et al. 2009). However, the technique has relatively low transfection efficiencies, especially in compared with other nonviral gene therapy techniques (Wells 2004; Kusumanto et al. 2007), which has hindered its development toward clinical application. In vivo administration of microbubbles is generally performed by injection into the blood supply either as a bolus or infusion. It has been shown in vitro that lipid-shelled microbubbles are susceptible to changes in pressure and shear stresses that occur during passage through syringes and needles (Barrack and Stride 2009). The concentration and size distribution of microbubble suspensions can be significantly decreased and altered, particularly when using large-gauge (small-bore) needles around 27–30 G (Talu et al. 2008). Few preclinical papers state the needle size used; however, because the preclinical model is typically a rodent, the needle size will usually be around 27–29 G (Sakakima et al. 2005; Wang et al. 2005). Considering the in vitro influence on microbubble size and concentration, there could be significant implications using such needles for UMGT. This may also contribute to the low transfection efficiencies often reported in vivo (Alete 2008; Wang et al. 2009; Phillips et al. 2010). This paper investigates the influence of needle gauge on transfection in vivo and presents simple methodological changes to improve the technique. MATERIALS AND METHODS Ultrasound contrast agent SonoVue (Bracco Research SA, Geneva, Switzerland) is a lipid-shelled microbubble of sulphur hexafluoride gas supplied as a lyophilized powder with 5 mL of saline for reconstitution. When prepared according to the manufacturer’s specification, the microbubble concentration ranges from 2–5 3 108 microbubbles/mL, with a mean diameter of 2.5 mm (Schneider 1999). In all experiments, SonoVue at double the normal concentration was used by reconstituting the lyophilized powder with half of the supplied saline because previous studies have shown that increasing the SonoVue concentration leads to
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increased transfection. However, increasing beyond double concentration leads to increased mortality in mice (Alete 2008). Plasmid The plasmid pGL4.13 (Promega UK Ltd., Southampton, UK) was used in all in vivo experiments. It carries the Phontinus pyraelis luciferase gene, luc2, controlled by the SV40 promoter and is 4.6 kb in size. It was transformed into a DH5a Escherichia coli strain following the manufacturer’s instructions (MAX Efficiency DH5a Competent Cells, Invitrogen Ltd., Paisley, UK). Plasmid was prepared from 2.4 L, overnight cultures using Qiagen’s EndoFree Gigaprep kit (Qiagen GmbH, Hilden, Germany) following the manufacturer’s instructions. Recovered plasmid was adjusted to a concentration of 4 mg/mL in injectable water for in vivo use. Syringes and needles Three gauges of needle were tested: a 25-G needle integrated in a 1-mL syringe (SurSaver syringe, Terumo Europe N.V., Leuven, Belgium, Cat. No.: SS01D2516); a 27-G needle integrated in a 0.5-mL syringe (Tuberculin syringe, BD Medical, NJ, USA, Cat. No.: 305620); and a 29-G needle integrated in a 0.5-mL syringe (Insulin syringe, Terumo UK Ltd., Surrey, UK, Cat. No.: BS 5 05M2913) (Table 1 has the full details). Integrated needles were used to minimize dead space, which is considerable when using standard hubmounted needles. This is an important consideration during microbubble administration because microbubbles float. This buoyancy is dependent on their gas volume, and therefore size, which can result in a heterogeneous size distribution in the syringe, developing over time. A portion of the dose is left in the dead space after administration, which leads to the loss of a size section of microbubble population. By using integrated needles, the dead space otherwise introduced by the needle-syringe connection is significantly reduced. In vitro UCA characterization In vitro samples were prepared to replicate the method used for in vivo delivery; 200 mL of a freshly prepared SonoVue suspension were removed to a 1.5-mL microcentrifuge tube using a 19-G needle and 1-mL
Table 1. Properties of the three syringes in use Syringe Terumo insulin syringe BD tuberculin syringe Terumo SurSaver syringe
Needle gauge 29 G 27 G 25 G
Needle ID (mm)
Needle OD (mm)
Needle length (mm)
Syringe volume (mL)
Syringe ID (mm)
Syringe OD (mm)
0.184 0.210 0.31
0.337 0.413 0.514
13.0 12.7 16.0
0.5 0.5 1.0
3.45 3.55 4.7
5.40 5.70 6.6
ID 5 Internal diameter; OD 5 outer diameter.
Influence of needle gauge on in vivo ultrasound d R. J. BROWNING et al.
syringe. One-hundred fifty mL of the suspension was added to 50 mL of injectable water by pipette and mixed gently by stirring the pipette tip. The 200-mL sample was drawn up using the experimental syringe and held vertically for 20 s as air bubbles were removed. The syringe was held horizontally for a further 20 s to simulate the placing of the needle, which would occur in vivo, and contents expelled into an empty microcentrifuge tube over 10 s. A ‘‘no-needle’’ control sample was also prepared by pipette from the diluted microbubble suspension. Samples were then diluted fivefold in water and placed on a hemocytometer for microscopy examination. After allowing 5 min for bubbles to rise to the cover-slip, 20 images were taken using a light microscope with digital acquisition (40x magnification, Nikon Eclipse 50i, Nikon Digital Camera DXM1200C, Nikon, Tokyo, Japan). Three sample repeats were prepared per syringe or control, with each sample imaged twice. Repeat samples were interleaved over time, to monitor the effect of bubble decay within the vial on suspension characteristics. Images were analyzed in MATLAB (The MathWorks Inc., Natick, MA, USA) using a custom sizing and counting program described in Sennoga et al. (2010) to determine the concentration, mean diameter, median diameter and gas concentration. Briefly, the program segments the bubbles from the image background. Individual bubbles are sized and counted. Clustered bubbles, identified through comparison with a circle, are excluded from the distribution estimation but included in the total bubble count. For further detail, please refer to Sennoga et al. (2010). The bubble decay, observed to be approximately linear over time, was calculated from control values and used to adjust our experimental results. In vivo transfection All animal work was conducted under the authority of a UK Home office project licence as required by the Animals (Scientific Procedures) Act 1986. Mice were maintained in a minimal-disease facility fully compliant with Home Office guidelines with food and water ad libitum. Six- to 8-week-old female CD1 mice were anesthetized using 5% isoflurane gas and maintained at 2–3% isoflurane on a heat mat. Hair was removed from the chest of each mouse by shaving and application of depilatory cream, to ensure good coupling with warmed ultrasound gel. A Siemens Acuson Sequoia (Siemens plc, Surrey, UK) ultrasound scanner and 15L8 linear array transducer were used throughout. Organ location and imaging was conducted at low intensity (14 MHz, 0.06 MI, focal depth 0.75 mm). The transducer was positioned using a cranialdorsal long-axis orientation through the left side of the chest so that the acoustic focus was coincident with the heart and acoustic emission frozen to prevent premature destruction of microbubbles. As described in the
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in vitro work, a 200-mL sample of microbubbles was removed from the SonoVue vial to a microcentrifuge tube using a 19-G needle hub mounted to a 1-mL syringe. A suspension of SonoVue (150 mL) and plasmid (50 mL) was prepared in a separate microcentrifuge tube and drawn into the experimental syringe. The syringe was held vertical for approximately 20 s as air bubbles were removed. Intravenous tail vein placement took an approximate further 20 s, during which time the syringe was held horizontally. The suspension was administered by hand at an approximate flow rate of 0.02–0.04 mL/s, i.e., each 200-mL injection took between 5 and 10 s. This was immediately followed by 2 min of high-intensity ultrasound (6 MHz, 1.6 MI, focal depth 0.75 mm) exposure, with the focus swept through the entire heart by slow movement of the transducer. After treatment, the transducer was removed and the mouse was recovered in a hot box. Bioluminescence imaging Three days after transfection, the transfected mice were imaged in vivo using an IVIS 100 bioluminescence camera and Living Image V3.2 software (Caliper Life Sciences, Hopkinton, MA, USA). Mice were anesthetized using 5% isoflurane and maintained at 1–2% isoflurane on the heated imaging platform of the IVIS 100 machine. An intraperitoneal injection of the substrate luciferin (Gold Biotechnology Inc., St. Louis, MO, USA) (300 mg/kg body weight in injectable water) was administered and the resulting bioluminescence in transfected tissues expressing luciferase was imaged. Bioluminescence images were then taken every two minutes (1-min exposure, field of view 10 cm, automatic height focusing) for 20 min. Using the analysis tools of the Living Image software, a region of interest was drawn around the area of bioluminescence and the photon flux (photon/s) measured. Signals were normalized for noise by removal of the background signal measured from an untreated site on each image. Statistics The in vivo data were determined to be normally distributed by the D’Agostino-Pearson K2 test and homoscedastic by Bartlett’s test. A one-way analysis of variance (ANOVA) was performed on the different needle groups to discern whether a statistical difference existed between the groups. Pairwise comparison was performed using Tukey’s test to determine which sample groups were significantly different. Significance was assumed if p # 0.05. Statistical analysis of in vitro data was limited to those bubbles acoustically active at clinical diagnostic frequencies (dia. 0.5 to 10 mm) (Gorce et al. 2000). In vitro needle and control data were compared using the derived
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population statistics of concentration, mean diameter, median diameter and gas concentration from each of the 6 repeats. The concentration, mean diameter and gas concentration were determined to be normally distributed and parametric statistical methods were used. Nonparametric methods were used to assess median diameters. All derived population statistics were found to be homoscedastic using Bartlett’s test or the BrownForsythe test. One way ANOVAs were used to determine whether a statistical difference existed between the needle groups in terms of the concentration, mean diameter and gas concentration. A Kruskal-Wallis ANOVA was used for median diameter. The parametric Tukey’s test or nonparametric Dunn’s test was used for pairwise comparison. Significance was assumed if p # 0.05. RESULTS Effect of needle gauge on transfection in vivo The in vivo peak bioluminescence measured three days after transfection was averaged over nine mice per needle group (see Fig. 1 for example images). A trend of increasing transfection with decreasing needle gauge is apparent (Fig. 2); using 27-G needles (7.21 3 105 photon/s) causes double the transfection compared with using 29-G needles (3.57 3 105 photon/s) while using 25-G needles (1.05 3 106 photon/s) significantly increases transfection almost threefold (p , 0.01). In addition, as the needle gauge increases so does the variability, with the standard deviation increasing between 29-G needles and 25-G needles; from 2.95 3 105 photon/s to 5.23 3 105 photon/s respectively. Effect of needle gauge on microbubbles in vitro To study the effect of needle gauge on microbubble suspensions directly, microbubbles were optically examined before and after passage through a needle (Fig. 3).
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Optical examination and comparative statistics revealed that, compared with the control, a 25-G needle caused no change in bubble concentration (Fig. 3a), mean diameter (Fig. 3b), median diameter (Fig. 3c) or gas concentration (Fig. 3d); a 27-G needle caused a slight (6%) drop in concentration and gas concentration but no change in the mean or median; a 29-G needle caused a significant drop in both concentration (30%) and gas concentration (45%), but only a slight decrease in mean diameter (9.8%) and median diameter (5.9%). In addition, 25-G and 27-G needles caused no change in the size distribution, whereas 29-G needles showed a downward shift in size distribution, because of the decrease in concentration.
DISCUSSION Our in vivo results demonstrate that using smaller needle sizes reduces the maximum transfection efficiency possible for a particular dose, a finding not reported before in the literature. This was supported by our in vitro work, which demonstrated a decrease in concentration as needle size is reduced, suggesting that a change in concentration is responsible for decreased transfection. The difference in concentration after needle passage between 29 G and 25 G supports the finding of a significant difference in transfection efficiency. However, the drop in concentration through a 27-G needle is more modest compared with the change in transfection. It is possible that additional factors may contribute to a lesser extent the differences observed in vivo, such as back pressure from the blood, or changes in blood flow caused by needle intrusion. In addition, it may be that needle passage affects the long term stability of a microbubble, a factor not investigated as part of this work. Work is currently underway to model and investigate these effects in vitro.
Fig. 1. Example in vivo bioluminescence images of mice transfected using (a) a 25-G needle, (b) a 27-G needle or (c) a 29-G needle. (Scale in photons per second per cm2 per steradian.).
Influence of needle gauge on in vivo ultrasound d R. J. BROWNING et al.
Fig. 2. In vivo peak bioluminescence levels three days after MBUS treatment averaged over nine mice per sample group. (Error lines are standard deviation, *p , 0.01 vs. mice treated with 29-G needles.).
Microbubble characteristics, such as size and concentration, play an important role in transfection. The size of a microbubble determines its resonant frequency and thus how it will respond to the acoustic driving frequency used during ultrasound exposure (Miller 1998; Chomas et al. 2001), which in turn affects the transfection efficiency (Chen et al. 2003). Several studies have demonstrated the link between microbubble suspension concentration
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and transfection (Koike et al. 2005; Alter et al. 2009). Because the bioeffects of cavitating microbubbles are thought to cause transfection, an increased number of microbubbles will cause increased bioeffects. This may also be related to a greater number of microbubbles being in contact with the cell, potentially leading to increased transfection. In addition, microbubbles in vitro tend to be more stable at higher concentrations (Feshitan et al. 2009), which may allow greater in vivo persistence and may increase the likelihood of sonoporation. There is a noticeable variability in each needle group tested. This is likely is related to limitations in the nature of the transfection and subsequent acquisition of the bioluminescence. Although the ultrasound exposure was focused to deliver maximum acoustic energy at a particular depth, propagation through the over- and underlying tissues is also likely to cause some unintentional plasmid transfection. Because the animals are biologically variable, e.g., in size and weight, some variability during transducer placement is unavoidable for the focus to be coincident with the heart. This could have led to a random distribution of transfection outside of the focus. Because the SV40 promoter on the plasmid is not tissue-specific, successful transfection in any tissue will lead to expression, which is detected by the
Fig. 3. Optically measured characteristics of microbubbles after needle passage. The plots show the mean value from six repeats per needle gauge adjusted for bubble decay measuring (a) concentration (microbubbles/mL), (b) mean diameter (mm), (c) median diameter (mm) and (d) gas concentration (mL/mL). Error bars show standard deviation per experimental syringe (*p , 0.05 vs. 29-G needle, yp , 0.05 vs. 25-G needle).
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bioluminescent camera. Indeed, ex vivo analysis of dissected thoracic tissues from transfected mice did in some cases reveal low levels of luminescence in the lungs and intercostal muscles. The range of injection time durations, from 5–10 s, could also have influenced the microbubble suspension finally delivered. Although our methodology aimed to replicate injection conditions for each mouse treated, even a small variation in administration time, in conjunction with bubble buoyancy and the shear stresses exerted on these bubbles close to the syringe wall during administration, could influence the suspension characteristics. These factors could therefore contribute to the variability seen for each group. However, this does not change the observation that an increased needle diameter has caused an increase in transfection. The in vitro investigation sought to identify potential reasons for the differences in transfection efficiency observed in vivo. From previous work conducted by Talu et al. (2008), we did not expect the flow rates and initial concentrations used would influence the microbubble populations before or after needle passage. We hypothesise that the syringe position and time taken to eliminate air bubbles (needle vertical for 20 s) and position of the needle for intravenous administration (needle horizontal for 20 s) alters the homogeneity of the suspension before delivery, and that this may influence both the number of bubbles affected by the shear stresses exerted at the syringe wall and the concentration of the suspension delivered. However, because the change in bubble population only occurs for the higher-gauge needles, the needle must be the primary agent in bubble destruction. It is possible that the shear stresses imposed on the bubbles as the plunger is depressed cause bubble instability. This instability leads to destruction during needle passage, but only if the needle is of sufficiently small diameter. It is likely that needle diameter is important because of the increase in pressure gradient from syringe barrel to needle or the greater shear stress occurring within the needle. It may be expected that larger bubbles rather than smaller would be more readily destroyed (Borden and Longo 2002; Kwan and Borden 2010); however, as previously noted, we limited our statistical analysis to clinically acoustic bubbles of 1–10 mm, and over this size range needle gauge did not cause a statistically significant alteration in microbubble size distribution or median diameter. It should be noted that there are important differences between our investigation and those conducted by Barrack and Stride (2009) and Talu et al. (2008). In the Barrack and Stride study, the needles used were detachable 18 G or 25 G attached to a 5-mL or 10-mL syringe depending on the experiment. The Talu et al. study instead used catheters of six-inch polyethylene tubing
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attached to needle gauges of 23 G, 27 G and 30 G. The diameter of the tubing matched the needle gauge in use and the syringe size was unspecified. It was therefore important to perform our own in vitro experiments to effectively examine the effect our syringes with integrated needles had on microbubble suspensions. Most importantly, our findings indicate that the largest needle size permitted by the preclinical model should be used when administering ultrasound contrast agent via intravenous injection. This will cause the least change to the microbubble population and their acoustic response and subsequent bioeffects. However, it must be noted that the vein diameter itself will limit the size of needle that can be used. In mice 6–8 weeks of age, a 25-G needle was the largest needle size that we judged could be safely used for intravenous injections. For larger animal models, this limit should be less significant. CONCLUSION We have shown here that plasmid transfection using ultrasound and the contrast agent SonoVue can be improved when using needles of a larger bore. UMGT has many advantages over other commonly used transfection techniques but is hampered by low transfection efficiency. Simple changes in the methodological procedures can help improve this by increasing the sensitivity of the system, which will allow observation of other optimization effects to be observed more clearly. Acknowledgments—This work was supported by an Engineering and Physical Sciences Research Council grant (EP/F066740/1).
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