Published online before print August 4, 2005, 10.1148/radiol.2371040805
(Radiology 2005;237:224-229.)
© RSNA, 2005
Gene Transfer with Microbubble Ultrasound and Plasmid DNA into Skeletal Muscle of Mice: Comparison between Commercially Available Microbubble Contrast Agents1
Xinghua Wang, MD2,
Hai-Dong Liang, PhD,
Baowei Dong, MD,
Qi-Long Lu, MD, PhD and
Martin J. K. Blomley, MD
1 From the Imaging Sciences Department (X.W., M.J.K.B.) and Muscle Cell Biology Group, Clinical Sciences Centre, Medical Research Council (Q.L.L.), Hammersmith Hospital, Imperial College London, Du Cane Rd, London W12 0NN, England; Imaging Sciences Department, 2nd Hospital, Shanxi Medical University, Taiyuan, China (X.W.); Department of Medical Physics and Bioengineering, Bristol General Hospital, Bristol, England (H.D.L.); Ultrasound Department, PLA General Hospital, Beijing, China (B.D.); and Neuromuscular/ALS Center, Carolinas Medical Center, Charlotte, NC (Q.L.L.). Received May 4, 2004; revision requested July 20; revision received November 15; accepted December 15. M.J.K.B. supported by U.K. Medical Research Council Career Establishment Grant G0100120.
Address correspondence to Q.L.L. (e-mail: qi.lu{at}carolinashealthcare.org).
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ABSTRACT
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PURPOSE: To compare three commercial microbubble contrast agents (Optison, SonoVue, and Levovist) for their effect on gene delivery in skeletal muscle in conjunction with the use of therapeutic ultrasound.
MATERIALS AND METHODS: The study was approved by the Animal Care and Use Committee. Plasmid DNA (10 µg) encoding green fluorescent protein (GFP) was mixed with microbubbles (or saline control) and injected into the tibialis anterior muscle of mice with and without adjunct ultrasound (1 MHz, 2 W/cm2, 30 seconds, 20% duty cycle). The efficiencies of GFP transgene expression were determined with four experimental conditions: (a) plasmid and saline as control (six mice), (b) plasmid and Optison (six mice), (c) plasmid and SonoVue (four mice), and (d) plasmid and Levovist (air based, four mice). The right legs were exposed to ultrasound, while the left legs were unexposed. Transfection efficiency was assessed by counting the number of GFP-positive fibers. Tissue damage was assessed by measuring the maximal-damage area on serial sections.
RESULTS: When ultrasound was applied, both SonoVue and Optison significantly improved (P < .05) gene transfection efficiency. Optison was also effective (P < .05) even when no ultrasound was applied, which is consistent with previous studies. Levovist without ultrasound decreased the level of transfection (P < .05), with increased tissue damage.
CONCLUSION: Both non–air-based agents show promise in gene delivery in skeletal muscle with undetectable tissue damage. Enhanced gene transfer with additional ultrasound was achieved only with SonoVue.
© RSNA, 2005
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INTRODUCTION
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Despite its great promise in treating a wide variety of diseases, safe and tissue-specific delivery remains arguably the biggest limitation to the wider clinical applications of gene therapy. Viruses are efficient as delivery systems but are associated with serious problems of immunogenicity and cytotoxicity (1). Gene delivery with plasmid DNA is safer, but the efficiency is poor (2); therefore, much effort has been made to develop new methods for improving its efficiency and tissue specificity. Microbubble ultrasound could fulfill such requirements. Ultrasound has been shown to enhance gene transfer, probably by the production of small and transient nonlethal pores in cell membranes (sonoporation) (3). Furthermore, targeted gene delivery could be achieved with the application of focused ultrasound.
Microbubbles, which are gas bubbles of around 3 µm in diameter, have been developed mainly as contrast agents to improve ultrasonographic (US) scans. However, they have shown promise in gene therapy for several reasons. Microbubbles act as cavitation nuclei, effectively focusing ultrasound energy, and can potentiate bioeffects (4), including sonoporation. There is evidence that the ultrasound energy needed can be greatly reduced, and therefore the lower power used in diagnostic imaging systems may be sufficient to produce therapeutic effects. In comparison, other physical methods, such as electroporation, can induce substantial tissue damage as a result of the powerful electric field required to achieve efficient gene delivery (5,6). Microbubbles can be targeted to tissues by incorporating ligands such as antibodies or peptides (7,8). A transgene can be either mixed with or complexed to microbubbles, thus increasing the probability of genes entering the cells (5,6).
Authors of several studies have also suggested that some microbubbles help enhance gene transduction in muscle (9–11). Muscle is an ideal target for studies of microbubbles and ultrasound-mediated gene delivery, because the tissue is easily accessible and naked DNA injection has but a limited effect on its own, which allows convenient comparison with experimental delivery strategies. In addition, muscle is a clinically relevant tissue to study, because gene therapy could be used both to treat devastating diseases such as Duchenne muscular dystrophy and for other purposes such as a "gene factory" (5,12). Study findings from our group showed that enhanced transgene expression could be achieved by using the contrast agent Optison (Amersham Health, Oslo, Norway) with direct injection (13). Findings of another study showed improved transfection by using the contrast agent Definity (Bristol Myers Squibb, St-Laurent, Quebec, Canada) (14).
Currently, several microbubble agents are commercially available for clinical imaging. The property of these microbubble agents varies in shell components, inner gas, size, and stability in biologic systems. Comparison of these microbubble agents for their effect on gene delivery could provide clues for further improvement of the technique and could help develop these methods for clinical applications, since the commercial microbubble agents are already licensed for patients use. Thus, the purpose of our study was to compare three commercial microbubble agents, Optison (licensed in the United States, Europe, and other areas), SonoVue (licensed in Europe and China), and Levovist (licensed in over 60 countries) for their effect on gene delivery in skeletal muscle in conjunction with the use of therapeutic ultrasound.
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MATERIALS AND METHODS
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Plasmid Preparations
A commercial reporter plasmid pEGFP (Clontech, Palo Alto, Calif) was used as a transgene in all experiments. The plasmid DNA was propagated from Escherichia coli DH5 cultures and was purified with kits (Qiagen Plasmid Maxi; Qiagen, Carlsbad, Calif) according to the company protocol. DNA concentration was determined with both ultraviolet spectrophotometry and agarose gel analysis.
Animals
Male C57Bl/10 mice (Hammersmith Hospital, London, England) between 4 and 5 weeks old were used in all experiments. The mice were divided into four groups; six mice each were used for the negative control (plasmid alone with no microbubbles) and Optison experiments, and four mice each were used for the SonoVue and Levovist experiments. Animals were anesthetized with an interperitoneal injection of 100 mL mixed solution (50% distilled water, 25% fentanyl and fluanisone, and 25% midazolam for each mouse; Janssen Animal Health, Beerse, Belgium). The body temperature of the mice was maintained at 37°C with a heating pad. The study was approved by the Animal Care and Use Committee at the Hammersmith Campus of Imperial College.
Microbubbles
Optison (Amersham Health, Amersham, UK) is an albumin-shelled US contrast agent composed of about 5–8 x 108 bubbles per milliliter that are between 2 and 4.5 µm in diameter and are filled with octaperfluoropropane. SonoVue (Bracco, Milan, Italy) is a lipid-shelled US contrast agent composed of millions of microbubbles filled with sulfur hexafluoride gas that are 2.5–6.0 µm in average diameter, and 90% of which are smaller than 6 µm. Levovist (Schering, Berlin, Germany) is a galactose-based air-filled microbubble contrast agent; 99% of the microbubbles are smaller than 7 µm. All contrast agents were prepared according to the manufacturer's instructions. Optison comes ready made and requires only resuspension, while SonoVue is reconstituted to 5 mg/mL concentration. Unlike with the other agents, the manufacturer data support the clinical use of several concentrations of Levovist (200–400 mg/mL). We used 200 mg/mL for all our experiments. In preliminary experiments, we confirmed that all the three microbubbles remain intact after passing through a 29-gauge needle when examined with a microscope.
Intramuscular Gene Delivery
In a total volume of 30 µL, 10 µg DNA of pEGFP expression vector was delivered into each tibialis anterior muscle by using 0.5-mL insulin syringes and 29-gauge needles (Becton Dickinson, Dublin, Ireland) by means of a single injection. Plasmid DNA was dissolved in 0.9% saline. When microbubbles were used, the same amount of plasmid DNA was dissolved in a total volume of 15 µL, and equal volume of microbubbles was added immediately before the injection. Control muscles were injected with plasmid DNA mixed with saline instead of microbubbles.
The efficiencies of GFP transgene expression were determined with four experimental conditions: (a) plasmid plus saline as control, (b) plasmid plus Optison, (c) plasmid plus SonoVue, and (d) plasmid plus Levovist. The right legs were exposed to ultrasound, while the left legs were unexposed. The procedure was performed by two authors (X.W., H.D.L.).
Ultrasound Exposure
A therapeutic ultrasound machine (Mark 3; EMS Limited, Oxford, England) was used. The frequency was 1 MHz, and the pulse repetition frequency was 100 Hz with a 20% duty cycle. The diameter of the probe was 35 mm, and the spatial peak temporal peak power level ranged 0.5–3.0 W/cm2. For the application of ultrasound on the plasmid-injected muscles, a tank containing water at room temperature and a platform placed just above the water surface were used. The mouse was placed on the platform, and the unilateral leg to be irradiated was immersed in the water; the other leg was not exposed to ultrasound. The insonation was performed about 1 minute after vector injection. For the ultrasound-exposed leg, the ultrasound probe was placed under water and was moved slowly around the area of tibialis anterior muscle in different directions. The entire treatment lasted 30 seconds.
Examination of GFP Expression in Muscles
Mice were sacrificed 1 week after plasmid DNA injection, since peak levels of transgene expression in muscle occur between 1 and 2 weeks in this model (15). The tibialis anterior muscles were removed and immediately snap frozen in isopentane cooled in liquid nitrogen. Sections 7 µm thick were cut at 100-µm intervals through whole tibialis anterior muscles and were mounted on glass slides. These sections were used either for fluorescence measurement or for muscle damage assessment. Fluorescence signals of GFP-positive fibers were examined by using fluorescence microscopy (Axiophot; Carl Zeiss, Gottingen, Germany), and images were captured by using confocal laser microscopy (LSM 5 PASCAL; Carl Zeiss, Welwyn Garden City, England). Readout was performed by three authors (X.W., H.D.L., Q.L.L.) using the section with the maximal number of fibers expressing GFP. The observers were blinded to the experimental group and conditions.
Muscle Damage Assessment
One set of sections from each sample was fixed with neutral buffered formalin, stained with hematoxylin and eosin, and examined with microscopy. The presence of centrally nucleated muscle fibers and foci of mononuclear infiltrates were used as signs of muscle damage. Muscle damage along the track of needle injection was traced microscopically, and the maximal area of damage on serial sections was measured and analyzed by one author (X.W.), who was supervised by a histologist (Q.L.L.) with more than 10 years of experience in muscle analysis. Because of the time involved in performing this assessment, we looked for damage in four mice from each group.
Statistical Analysis
Data are expressed as means and standard deviations. All comparisons were performed for the number of transfected fibers and the area of damage. We separately examined differences between the microbubbles and between samples with and without ultrasound. We used nonparametric tests, because a Bartlett test for homogeneity of variances suggested significant differences in the standard deviations of the groups.
A Kruskal-Wallis analysis of variance test was first used, and two-column comparisons were made. If a significant difference was seen, groups of mice treated with microbubbles were compared with the negative control group (ie, plasmid with no microbubbles) by using a two-tailed nonparametric comparison (Mann-Whitney U test). Because three comparisons were possible, a Bonferroni correction was performed by multiplying the probability value by three. We also compared each group for the effects with and without ultrasound. We did this by using nonparametric two-column comparisons between studies with and studies without ultrasound for each experimental condition. Differences were considered significant at P < .05. The software packages used were SPSS, version 11.5 (SPSS, Chicago, Ill), and Instat 2.0 (GraphPad Software, San Diego, Calif).
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RESULTS
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Optison Improves Transgene Expression without Ultrasound
In the absence of ultrasound, a significant difference (P < .01, Kruskal-Wallis test) was observed among the groups. When Optison alone was used, there was a significant (76.8 ± 29.2 vs 17.7 ± 3.8; P < .01, Mann-Whitney U test, Bonferroni correction) increase in the number of GFP-positive fibers compared with that in the negative control group. SonoVue alone showed a weak but not significant trend to improve transgene expression (P = .057, Mann-Whitney U test, Bonferroni correction). Levovist alone, however, significantly decreased the transfection efficiency when compared with that in the negative control group (7.0 ± 5.2 vs 17.7 ± 3.8; P < .05, Mann-Whitney U test, Bonferroni correction) (Figs 1, 2).
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Figure 1. Bar graph depicts number of GFP-positive fibers in mouse skeletal muscle. Data are mean ± standard deviation (n = 4 or 6). * = Comparison across groups with negative control (saline) in the absence of ultrasound, with P < .05; # = comparison across groups with negative control (saline) in the presence of ultrasound, with P < .05.
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Figure 2a. Phase-contrast confocal microscopy images show GFP-positive fibers emitting green fluorescence signals. With (a) saline, two GFP-positive fibers are visible; with (b) saline plus ultrasound, one GFP-positive fiber is visible; and with (c) Optison, (d) Optison plus ultrasound, (e) SonoVue, and (f) SonoVue plus ultrasound, more GFP-positive fibers are visible. (g) With Levovist and (h) Levovist plus ultrasound, the signal of GFP-positive fibers was too weak to be displayed. (Original magnification, x20.)
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Figure 2b. Phase-contrast confocal microscopy images show GFP-positive fibers emitting green fluorescence signals. With (a) saline, two GFP-positive fibers are visible; with (b) saline plus ultrasound, one GFP-positive fiber is visible; and with (c) Optison, (d) Optison plus ultrasound, (e) SonoVue, and (f) SonoVue plus ultrasound, more GFP-positive fibers are visible. (g) With Levovist and (h) Levovist plus ultrasound, the signal of GFP-positive fibers was too weak to be displayed. (Original magnification, x20.)
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Figure 2c. Phase-contrast confocal microscopy images show GFP-positive fibers emitting green fluorescence signals. With (a) saline, two GFP-positive fibers are visible; with (b) saline plus ultrasound, one GFP-positive fiber is visible; and with (c) Optison, (d) Optison plus ultrasound, (e) SonoVue, and (f) SonoVue plus ultrasound, more GFP-positive fibers are visible. (g) With Levovist and (h) Levovist plus ultrasound, the signal of GFP-positive fibers was too weak to be displayed. (Original magnification, x20.)
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Figure 2d. Phase-contrast confocal microscopy images show GFP-positive fibers emitting green fluorescence signals. With (a) saline, two GFP-positive fibers are visible; with (b) saline plus ultrasound, one GFP-positive fiber is visible; and with (c) Optison, (d) Optison plus ultrasound, (e) SonoVue, and (f) SonoVue plus ultrasound, more GFP-positive fibers are visible. (g) With Levovist and (h) Levovist plus ultrasound, the signal of GFP-positive fibers was too weak to be displayed. (Original magnification, x20.)
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Figure 2e. Phase-contrast confocal microscopy images show GFP-positive fibers emitting green fluorescence signals. With (a) saline, two GFP-positive fibers are visible; with (b) saline plus ultrasound, one GFP-positive fiber is visible; and with (c) Optison, (d) Optison plus ultrasound, (e) SonoVue, and (f) SonoVue plus ultrasound, more GFP-positive fibers are visible. (g) With Levovist and (h) Levovist plus ultrasound, the signal of GFP-positive fibers was too weak to be displayed. (Original magnification, x20.)
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Figure 2f. Phase-contrast confocal microscopy images show GFP-positive fibers emitting green fluorescence signals. With (a) saline, two GFP-positive fibers are visible; with (b) saline plus ultrasound, one GFP-positive fiber is visible; and with (c) Optison, (d) Optison plus ultrasound, (e) SonoVue, and (f) SonoVue plus ultrasound, more GFP-positive fibers are visible. (g) With Levovist and (h) Levovist plus ultrasound, the signal of GFP-positive fibers was too weak to be displayed. (Original magnification, x20.)
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Figure 2g. Phase-contrast confocal microscopy images show GFP-positive fibers emitting green fluorescence signals. With (a) saline, two GFP-positive fibers are visible; with (b) saline plus ultrasound, one GFP-positive fiber is visible; and with (c) Optison, (d) Optison plus ultrasound, (e) SonoVue, and (f) SonoVue plus ultrasound, more GFP-positive fibers are visible. (g) With Levovist and (h) Levovist plus ultrasound, the signal of GFP-positive fibers was too weak to be displayed. (Original magnification, x20.)
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Figure 2h. Phase-contrast confocal microscopy images show GFP-positive fibers emitting green fluorescence signals. With (a) saline, two GFP-positive fibers are visible; with (b) saline plus ultrasound, one GFP-positive fiber is visible; and with (c) Optison, (d) Optison plus ultrasound, (e) SonoVue, and (f) SonoVue plus ultrasound, more GFP-positive fibers are visible. (g) With Levovist and (h) Levovist plus ultrasound, the signal of GFP-positive fibers was too weak to be displayed. (Original magnification, x20.)
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Ultrasound Improves Efficiency When Used in Combination with SonoVue
In the presence of ultrasound, a significant difference (74.5 ± 24.8 vs 20.3 ± 7.1; P < .01, Mann-Whitney U test, Bonferroni correction) was observed in the number of GFP-positive fibers between Optison plus ultrasound and plasmid plus ultrasound groups. However, there was no significant difference between Optison plus ultrasound and Optison alone groups. The use of ultrasound significantly improved the efficiency of transgene expression in the presence of SonoVue (66.0 ± 14.8 vs 20.3 ± 7.1; P < .05, Mann-Whitney U test, Bonferroni correction), with the number of GFP-positive fibers comparable to those in the Optison plus ultrasound group. However, the combination of Levovist and ultrasound remained ineffective, with no significant difference (n = 4 or 6, Mann-Whitney U test, Bonferroni correction) in the number of GFP-positive fibers compared with that in the plasmid plus ultrasound group (Figs 1, 2).
Tissue Damage Assessment
To assess the possible cytotoxic effect on muscle of the microbubbles with or without ultrasound irradiation, the maximal area of damage was determined according to the presence of centrally nucleated fibers (indication of muscle regeneration after damage) and necrosis. Plasmid only produced small areas of muscle damage along the needle tracks with or without ultrasound. There was no significant difference (n = 4, Mann-Whitney U test, Bonferroni correction) in muscle damage between plasmid and Optison and SonoVue groups with and without ultrasound. Levovist with or without ultrasound showed a trend of increased tissue damage when compared with that in all other groups, including saline control, but the trend was not significant (n = 4, Mann-Whitney U test, Bonferroni correction) (Tables 1,2).
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DISCUSSION
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Three commercial microbubble agents (Optison, SonoVue, and Levovist) with and without ultrasound were compared in their ability to improve the efficiency of plasmid DNA expression in skeletal muscle. SonoVue combined with ultrasound significantly improved the transgene expression in skeletal muscle without increasing tissue damage at microscopic level. Optison, but not SonoVue, was effective without ultrasound. However, Levovist with or without ultrasound did not show any benefit; instead, it increased tissue damage.
While we were finishing our experiments, Li et al (16) reported a similar study in which different microbubbles were compared for their effect on gene transfer in skeletal muscles. Findings of both studies demonstrate that Optison improves gene expression in conjunction with ultrasound and that the use of Levovist with or without ultrasound had no effect on gene transfection. However, our results differ from their results with regard to the effect of Optison. Li et al reported that there was no effect on transgene expression when Optison was used alone. However, our study findings, which are consistent with our previous results, showed that Optison improves gene expression significantly when compared with plasmid only. In fact, there is no significant difference in the number of GFP-positive fibers between Optison alone and Optison plus ultrasound, which suggests that ultrasound did not further improve the transfection efficiency obtained with Optison.
It is not clear why we observed an effect with Optison alone while Li et al (16) did not. However, in our previous study, we noted that changing the age of the mice being studied was important (the effect of Optison alone being much lower in older mice). It may therefore be that differences in the mouse strain or age are at least a partial explanation: We used 4-week-old C57Bl/10 mice, while Li et al used 6-week-old BALB/C mice. In addition, we believe, we have performed the first evaluation of SonoVue for this application. SonoVue with ultrasound showed a significant effect on gene transfection, but no convincing effect was seen without ultrasound. This suggests that the major effect of SonoVue is to potentiate the sonoporation effect of ultrasound. In contrast, Optison appears to have an innate effect, which is perhaps related to its chemical composition and gas or shell contents (Optison has an albumin shell and an octafluoropropane content, while SonoVue is made from a phospholipid shell with sulfur hexafluoride gas and Levovist is galactose- and air-based contrast agent). This effect is sufficiently large to dominate over any effects due to the potentiation of ultrasound.
We do not know which component of Optison is important. There have been some experiments that showed viral protein (17) or cationized albumin (18) and cationic lipids (19–21) could or had the potential to improve transgene expression. It is, therefore, possible that albumin or the lipid itself might contribute to the improved gene delivery in muscles. This is supported by the report (14) that the use of microbubble agent Definity, which is also shelled by lipid, had a significant effect on transgene expression in mice muscles. The perfluorocarbon gases used in both Optison and Definity may also be important. The lower solubility of the perfluorocarbon gas–filled agents provides higher stability (22) than do air-filled agents, which might be more important to the process of sonoporation. This hypothesis is supported by the fact that significant enhancement of gene transfer was observed only with the perfluorocarbon gas–filled microbubbles Optison and SonoVue but not with the air-filled microbubble Levovist. It is therefore likely that perfluorocarbon gas–filled microbubbles form more bubbles, serving as cavitation nuclei locally, than do air-filled microbubbles when injected into tissues in vivo. The increased number of bubbles with Optison and SonoVue therefore can lower the cavitation threshold, with an increased sonoporation effect on cell membrane, thus resulting in the higher efficiency of transgene delivery.
On the other hand, we noted that Levovist increased muscle damage, which might be another reason for the disappointing effects with this agent. Levovist is known to be a hypertonic agent, and we suspect that the hypertonicity of the galactose solution is the main mechanism. It is also possible that galactose and palmitic acid, added as a stabilizer, might contribute to tissue damage. Some authors (23) observed that Levovist could induce early apoptosis and noted that the effect could also be produced by using the same concentration of galactose. Furthermore, those authors (23) observed higher levels of secondary necrosis with Levovist than with the same concentration of galactose. They hypothesized that these findings might be a result of the presence of palmitic acid.
Our study had some limitations that mainly related to the need for more studies to investigate the hypotheses generated as a result of this study. We decided to focus initially on a relatively simple model with which we have extensive experience and in which we had a baseline for transduction efficiency. Ideally, it would obviously have been preferable to study more animals at different time points and of different ages. This was not possible owing mainly to the large amount of time needed to get a histologic readout, but in the future we plan to study serial expression more easily and noninvasively by using a newly acquired bioluminescence camera and luciferase reporter. Although no beneficial effect was seen with Levovist, it may be that a small effect is present and would have been seen in a larger study. However, even with the numbers studied, our data point to the non–air-based agents as being more effective and the ones to focus on for future studies.
It is important to understand the life span of microbubbles in tissue with ultrasound; unfortunately, no method able to image microbubbles selectively at the high frequency needed was available at the time we performed these experiments. We would have liked to have been able to standardize microbubble numbers so the effects of identical numbers of admixed microbubbles could be compared, but this was not possible owing to the differences in the way manufacturers list concentration (Optison is listed as having 5–8 x 108 particles per milliliter, while the other agents are listed in milligrams per milliliter). In addition, microbubbles have a wide range of sizes, and the distribution and/or size profile varies between agents, which will further confound this sort of comparison. Finally, the actual importance of the increases seen in therapeutic gene transfer is unknown and merits further investigation.
Commercially available licensed microbubbles show promise in potentiating gene therapy. These methods would need to be developed further but may be feasible for clinical applications. In this regard, newer microbubbles with lower solubility gases are better suited for gene delivery than are air-based microbubbles (eg, Levovist). Both Optison and SonoVue did not show any detrimental effect on muscle tissue. In contrast, Levovist increases muscle damage without improving efficiency for gene delivery when used either alone or in combination with ultrasound. These results therefore suggest that improvements in the chemistry and composition of a microbubble may enable us to develop more potent gene delivery vehicles with minimum side effects.
Microbubble ultrasound enhanced gene transfer while reducing tissue damage; hence, it may provide a safe and effective method for enhancing gene transfer to dystrophic muscles, thereby increasing the prospects for therapeutic application of naked DNA in muscular dystrophy and for other applications.
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FOOTNOTES
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Abbreviations: GFP = green fluorescent protein
2 Current address: Imaging Sciences Department, 2nd Hospital, Shanxi Medical University, Taiyuan, China 
Authors stated no financial relationship to disclose.
Author contributions: Guarantors of integrity of entire study, all authors; study concepts, all authors; study design, X.W., H.D.L., M.J.K.B.; literature research, X.W.; experimental studies, H.W., H.D.L.; data acquisition, X.W.; data analysis/interpretation, X.W., Q.L.L., M.J.K.B.; statistical analysis, X.W., M.B.; manuscript preparation, X.W.; manuscript definition of intellectual content and editing, all authors; manuscript revision/review, H.D.L., Q.L.L., M.J.K.B.; manuscript final version approval, all authors
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