Improving DNA vaccination performance through a new microbubble design and an optimized sonoporation protocol

Graphical abstract

Highlights

• •A new microbubble design is provided for maximizing the DNA delivery efficiency of cavitation-induced sonoporation.
• •An optimized sonoporation protocol effectively improved the vaccination performance of a DNA vaccine.
• •Whole genome resequencing demonstrated for the first time the safety of sonoporation in mediating DNA delivery.

Abstract

As a non-viral transfection method, ultrasound and microbubble-induced sonoporation can achieve spatially targeted gene delivery with synergistic immunostimulatory effects. Here, we report for the first time the application of sonoporation for improving DNA vaccination performance. This study developed a new microbubble design with nanoscale DNA/PEI complexes loaded onto cationic microbubbles to attain significant increases in DNA-loading capacity (0.25 pg per microbubble) and in vitro transfection efficiency. Using live-cell imaging, we revealed the membrane perforation and cellular delivery characteristics of sonoporation. Using luciferase reporter gene for in vivo transfection, we showed that sonoporation increased the transfection efficiency by 40.9-fold when compared with intramuscular injection. Moreover, we comprehensively optimized the sonoporation protocol and further increased the transfection efficiency by 43.6-fold. Immunofluorescent staining results showed that sonoporation effectively activated the MHC-II⁺ immune cells. Using a hepatitis B DNA vaccine, sonoporation induced significantly higher serum antibody levels when compared with intramuscular injection, and the antibodies sustained for 56 weeks. In addition, we recorded the longest reported expression period (400 days) of the sonoporation-delivered gene. Whole genome resequencing confirmed that the gene with stable expression existed in an extrachromosomal state without integration. Our results demonstrated the potential of sonoporation for efficient and safe DNA vaccination.

1. Introduction

During the past two decades, outbreaks of newly emerged viruses, such as the severe acute respiratory syndrome coronavirus (SARS-CoV) outbreak that began in 2003, the H1N1 avian influenza outbreak that began in 2009, the Middle East respiratory syndrome coronavirus (MERS-CoV) outbreak that began in 2012, the Zika virus outbreak that began in 2016, and the SARS-CoV-2 (also known as COVID-19) outbreak that began in 2019, have greatly affected the physical and mental health of the global population [1]. Vaccination has been widely accepted as the most effective medical intervention against infectious viral diseases. For example, it has been estimated that by April 10, 2021 Israel averted 79.1 % of COVID-19-related hospitalizations and 79 % of deaths via its nationwide vaccination campaign that started on December 20, 2020 [2]. In the global efforts toward COVID-19 vaccine development, nucleic acid vaccines have demonstrated their advantages of a short development cycle, rapid manufacture, and safe application [3], [4]. Two mRNA vaccines (by Pfizer-BioNTech and Moderna) have achieved high protection efficacy against COVID-19 (95 % and 94.5 %, respectively); however, the global application of mRNA vaccines is still hindered by the demanding storage temperatures and relatively high nano-encapsulation costs of these vaccines [5].

Compared with mRNA vaccines, DNA vaccines are more stable in storage, more resistant to nuclease degradation, and less expensive to produce [4]. In August 2021, the government of India granted Emergency Use Authorization to a COVID-19 DNA vaccine (ZyCoV-D). Additionally, a variety of DNA vaccines to fight against disease-causing viruses, such as MERS-CoV [6], avian influenza virus H7N9 [7], Zika virus [8], and human immunodeficiency virus (HIV) [9], have entered human clinical trials. Notably, the antigen expression efficiency of DNA vaccines is lower than that of mRNA vaccines because DNA plasmids must bypass one more cellular barrier (i.e., the nuclear membrane) to initiate the gene transfection and expression [10]. Insufficient intracellular delivery of a DNA vaccine leads to an inability to produce enough viral antigens to effectively activate the host immune system [11]. Therefore, various physical methods have been developed to assist in the intracellular delivery of DNA vaccines, such as electroporation [6], [12] and high-pressure jet injection [7], [8], [9]. Here, we for the first time applied the technique of sonoporation to improve the transfection and immunization of a DNA vaccine against hepatitis B virus (HBV).

Sonoporation refers to the biophysical process that the ultrasound-driven oscillation or collapse of microbubbles (gas-filled micro-spheres of 1–5 μm in diameter), which is also known as ultrasonic cavitation, mechanically perforates the nearby cell membrane [13], [14]. The sonoporation-created cell membrane perforation with a diameter ranging from tens of nanometers to several micrometers can deliver drugs and genes into the cytoplasm [15], [16]. Owing to the spatial focusing ability of ultrasound energy, sonoporation can achieve non-invasive and target-specific transfection with a resolution of 1–2 mm in deep tissues [17], [18], [19], [20]. Therefore, sonoporation-mediated intracellular delivery has been applied to gene therapy for brain diseases [21], cardiovascular diseases [22], [23], bone fracture [24], and tumors [25]. However, sonoporation has not yet been explored for DNA vaccination. Moreover, it is noteworthy that sonoporation itself can serve as a potent physical adjuvant for immunostimulation and immunomodulation [26], [27]. It has been found that sonoporation could induce sterile inflammation responses by elevating the local expression of pro-inflammation factors (e.g., heat shock protein 70, IL-1, IL-18, and TNF-α) during intracellular delivery [28]. Here, we propose the use of sonoporation to improve the immunization outcomes of DNA vaccines because sonoporation can increase transfection efficiency and simultaneously enhance immune responses.

It has been reported that the intracellular delivery efficiency of sonoporation decreased with the increasing molecular weight of model drugs [29]. Compared with typical drugs, which generally have small molecular weights (<1 kDa), the molecular weights of DNA vaccines are much larger (>1,000 kDa) [30]. Therefore, cationic polymers have been used in sonoporation to condense the negatively-charged DNA plasmids into smaller nanoparticles/complexes for higher delivery efficiency [31]. By adding polyethylenimine (PEI) into the extracellular medium with DNA plasmids, the in vitro cell transfection efficiency increased with an increasing ratio of PEI nitrogen to DNA phosphate (N:P ratio) [32]. However, for in vivo gene transfection, the inherent toxicity of PEI necessitated the use of targeted delivery to reduce systemic toxicity. Therefore, PEI was hybridized with the lipid shell of the microbubbles to effectively load DNA and to achieve targeted transfection in the focal spot of ultrasound [33]. Positively-charged lipids (e.g., DMAPAP, DOTMA and DOTAP) were also added to the microbubble shell to achieve targeted transfection [19], [34]. Cationic microbubbles resulted in a higher transfection efficiency compared with that of neutral microbubbles. However, the DNA-loading ability of cationic microbubbles was reported to be relatively low (0.02–0.03 pg per microbubble) [35], [36].

In this study, we designed a new microbubble structure to increase the total amount of the DNA vaccine loaded on the microbubbles. More specifically, we first used PEI to condense the DNA vaccine into nanoscale complexes (Fig. 1A) and then electrostatically loaded the DNA/PEI complexes onto cationic microbubbles to attain a higher DNA-loading capacity (Fig. 1B). When an ultrasound is used to drive the oscillation or collapse of microbubbles, the mechanical effects generated by the microbubbles can disrupt the cell plasma membrane and deliver DNA vaccine with an enhanced transfection efficiency (Fig. 1C). The expression of the virus antigen (e.g., hepatitis B surface antigen (HBsAg)) can then attract antigen-presenting immune cells to trigger immune responses. Furthermore, we comprehensively optimized the sonoporation parameters for intramuscular transfection, including acoustic pressure of the ultrasound, N/P ratio of the DNA/PEI complex, concentration of microbubbles, the number of split doses, and the time interval of repeated transfection. To examine the immunization effect of the DNA vaccine, we investigated the accumulation of antigen-presenting immune cells at the sonoporation site and examined the temporal profile of antibody production. In addition, we carried out whole genome resequencing of the mice with stable long-term (400 days) gene expression to identify potential DNA insertions and to examine the safety of sonoporation-mediated DNA vaccination.

Fig. 1.

Design and characterization of DNA/PEI complex-loaded microbubbles. (A–B) Graphic illustrations of the electrostatic formation of DNA/PEI complexes (A) and DNA/PEI complex-loaded microbubbles (B). (C) Graphic illustration of the transfection of muscle cells with DNA vaccine using sonoporation. (D) Gel electrophoresis of the HBsAg DNA vaccine. (E–F) The measured hydrodynamic size (E) and zeta potential (F) of DNA/PEI complexes with different N:P ratios. (G) Zeta potential of complex-loaded microbubbles with different N:P ratios. (H) Bright-field (BF) and fluorescent microscopy images of neutral and cationic microbubbles co-incubated with DNA/PEI complexes. (I) Gel electrophoresis of the HBsAg DNA vaccine mixed with different amounts of cationic microbubbles. Scale bar: 10 μm. PI: propidium iodide. MB: microbubble.

2. Materials and methods

2.1. DNA vaccine and reporter plasmids

To construct a DNA vaccine against HBV, the cloned genes expressing the HBV proteins preS2 and S were inserted into the pcDNA™ 3.1(+) vector, which has a human cytomegalovirus (CMV) immediate-early promoter. Reporter plasmids expressing enhanced green fluorescence protein (EGFP) and luciferase were constructed using the same method. All plasmids were expanded in Escherichia coli and extracted using endotoxin-free mass extraction kits (Omega Bio-tek, Norcross, GA, USA). The concentration of the extracted plasmids was measured using a UV–vis Spectrophotometer (Nanodrop 2000, Thermo Scientific, Wilmington, DE, USA). Agar gel electrophoresis was carried out to ensure the quality of the plasmids.

2.2. DNA/PEI complexes and DNA/PEI complex-loaded microbubbles

DNA plasmids prepared as described above were mixed with branched PEI (25 kDa, Sigma-Aldrich, St. Louis, MO, USA) at an N/P ratio of 0.5, 3, or 7. The mixture was vortexed for 10 s and then incubated at 4 ℃ for 30 min. Cationic microbubbles (USphere™ Trans⁺) containing DOTAP lipids were purchased from TRUST Bio-sonics (Taiwan, China). The DNA/PEI complexes were added into cationic microbubble solutions of varying concentrations (2 × 10⁶–1 × 10⁸ microbubbles/mL) and incubated at 4 ℃ for 10 min to generate DNA/PEI complex-loaded microbubbles. Both the DNA/PEI complexes and the complex-loaded microbubbles were prepared in phosphate-buffered saline (PBS) buffer (pH = 7.4).

2.3. Characterization of DNA/PEI complexes and DNA/PEI complex-loaded microbubbles

The hydrodynamic diameters and zeta potentials of DNA/PEI complexes with different N/P ratios were measured using a Zetasizer Nano instrument (Malvern Panalytical, Malvern, UK). The zeta potentials of the cationic microbubbles and DNA/PEI complex-loaded microbubbles were measured using the same instrument. To label the DNA that was loaded on the microbubbles, the DNA/PEI complex-loaded microbubbles were incubated with the fluorescence dye propidium iodide (PI) (0.1 mg/mL, 5 min, 4 °C), washed three times with cold PBS buffer, and centrifuged (300 RCF, 10 min, 4 °C) for collection. The fluorescently labelled DNA and the microbubbles were imaged using a Nikon C2 + confocal laser scanning microscope (Nikon Instruments, Melville, NY, USA).

2.4. Cell culture and microscopic imaging

Human fibroblast cells (MRC-5) were cultured in Minimum Eagle’s Medium supplemented with 10 % fetal bovine serum. Upon reaching 70 % confluency, the cells were subcultured and seeded in customized culture dishes with bilayer experiment windows for inducing sonoporation [15]. The cell plasma membrane was labeled with CellMask™ Orange (catalog No.: C10045, Thermo Fisher Scientific, Waltham, MA, USA) to observe the perforation and recovery process after sonoporation. The DNA plasmids were covalently labelled with far-red-fluorescent Label IT™ Cy5 dye (Mirus Bio, Madison, WI, USA) to track their cellular delivery. Both the sonoporation of the cell plasma membrane and the cellular delivery of DNA plasmids were imaged using a ZEISS LSM880 confocal microscope (Zeiss, Jena, Germany).

2.5. Animals

Female BALB/c mice were purchased from the Guangdong Medical Laboratory Animal Center (Foshan, Guangdong, China). The animals were maintained in a specific-pathogen-free environment. The protocols for the in vivo gene transfection experiments were approved by the Animal Ethical and Welfare Committee of Health Science Center of Shenzhen University. Before the transfection experiments, the hair on the hind legs of 6-week-old mice was removed using an electric trimming machine and depilatory cream. During the transfection experiments, the animals were anesthetized with 2 % isoflurane gas.

2.6. Ultrasound exposure

Ultrasound parameters, including peak negative pressure (PNP), pulse duration (PD), pulse repetition frequency (PRF), and exposure time (T), were edited in a signal generator (33512B, Keysight Technologies, Santa Clara, CA, USA). The generated signal was amplified by a 53-dB power amplifier (2100L, Electronics & Innovation, Rochester, NY, USA) and transformed into ultrasound by a 0.5-MHz single element transducer (I8-0018P-SU, Olympus NDT, Waltham, MA, USA). The ultrasound energy used for inducing sonoporation was measured using a needle hydrophone (model: HNR-0500, Onda, Sunnyvale, CA, USA). In transfection experiments, live cells or the hind legs of the animals were placed in the focal point of the ultrasound field (68 mm away from the front of the transducer) to receive ultrasound exposure. The measured lateral 6-dB diameter of the ultrasound beam was 16 mm. Note that ultrasound pressures of 0.075–0.8 MPa have been used for in vivo gene transfection [19], [22], [25]. However, Xie et al. reported that hemorrhagic bioeffects in the muscle tissues could be avoided in sonoporation when ultrasound pressure was set to be lower than 0.6 MPa [23]. Therefore, we selected ultrasound pressures of 0.13, 0.28, 0.43, and 0.58 MPa for inducing cavitation of different intensities.

2.7. In vitro quantification of EGFP reporter plasmid expression

DNA/PEI complex-loaded microbubbles were added into the customized culture dishes with a cell-to-microbubble ratio of 1:2. Then, the customized culture dishes were flipped upside down to receive ultrasound exposure, which facilitated the contact of the cells and microbubbles with the aid of buoyant forces. For each sonoporation group, three independent transfection experiments (three culture dishes for each experiment) were conducted. Cells in the center of culture dishes were imaged for EGFP expression 24 h after sonoporation. Total numbers of 1200–1800 cells in each sonoporation group were counted manually for the quantification of EGFP expression.

2.8. In vivo imaging of luciferase reporter plasmid expression

Anesthetized mice were injected intraperitoneally with D-luciferin (15 mg/mL in PBS, 10 µL per gram of bodyweight). The animals were then imaged using quantitative bioluminescent imaging equipment (IVIS lumina Ⅱ, Caliper Life Sciences, Hopkinton, MA, USA) 15 min after the injection of D-luciferin. The exposure time of the IVIS lumina Ⅱ equipment was set to be 1 min, and the f/stop aperture was 4.

2.9. Enzyme-linked immunosorbent assay (ELISA) detection of anti-HBsAg antibody

Blood samples (50 μL) were collected from the orbital venous sinus of the anesthetized mice at various time points (2–56 weeks) after the last vaccination. After being left to clot at room temperature for 1 h, the samples were centrifuged at 1,000 × g for 10 min to extract the serum. To reduce the matrix effect, the extracted serum was diluted (1:8). The concentration of anti-HBsAg antibody in the diluted serum was measured using an ELISA kit (Renjie Biological Technology Corp., Shanghai, China) with antibody standards ranging from 0 to 8 ng/mL.

2.10. Histological staining of muscle slices

After the sacrifice of the mice, muscle tissues were first excised using surgical scissors and then fixed in 4 % paraformaldehyde solution for 24 h. To investigate the sonoporation-induced muscle damage, muscle slices (5-µm thickness) were stained with Masson-trichrome staining. For immunohistochemical examination of the sonoporated muscles, muscle slices were stained with an anti-caspase-3 recombinant monoclonal antibody (catalog No.: 700182). To immunofluorescently label the expressed HBsAg and the histocompatibility complex class Ⅱ (MHC Ⅱ) molecules, muscle slices were stained with an anti-HBV surface Ad/Ay polyclonal antibody (catalog No.: PA1-73084) and an anti-MHC class Ⅱ monoclonal antibody (catalog No.: 14–5321-82). All the antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Assay-dependent dilution of antibodies ranged from 1:500 to 1:2000.

2.11. Whole-genome resequencing

Total genomic DNA was extracted from the leg muscles (n = 6) of the mice with sonoporation-mediated gene transfection and persistent luciferase expression. A DNA library (insert size of 500 bp) was constructed using the Hieff next-generation sequencing (NGS) MaxUp II DNA library Prep Kit (12200ES08, YEASEN Biotech, shanghai, China). Library fragments were purified with Hieff NGS™ DNA Selection Beads (12601ES56, YEASEN Biotech, shanghai, China). The concentration of the DNA library was quantified on a Qubit 4.0 fluorometer (Q33238, Thermo Fisher Scientific, Waltham, MA, USA) with the Qubit dsDNA HS Assay Kit (Q32854, Thermo Fisher Scientific, Waltham, MA, USA). Whole genome sequencing was performed on the MGI DNBSEQ-T7 platform (MGI-TECH, ShenZhen, China).

2.12. Statistical analysis

All statistical analysis was carried out with GraphPad Prism (version 8.3.0, GraphPad Software, San Diego, CA). Data are expressed as mean ± standard deviation (SD). Statistical significance was determined using the unpaired t test with Welch's correction and without assuming equal SDs.

3. Results

3.1. Characterization of the DNA/PEI complex-loaded microbubbles

A representative electrophoresis gel of the HBsAg DNA vaccine is displayed in Fig. 1D, showing that the vaccine is mainly composed of supercoiled DNA with high transfection efficiency (bottom band). The mean zeta potential and hydrodynamic diameter of the HBsAg DNA vaccine were measured to be − 65.9 ± 1.4 mV and 941.1 ± 43.0 nm, respectively (Fig. 1E & F). When the DNA vaccine was condensed by PEI to form DNA/PEI complexes, the mean hydrodynamic diameters of the resulting complexes with N/P ratios of 0.5, 3, or 7 were 263.0 ± 11.2, 384.4 ± 31.4, or 507.7 ± 43.5 nm, respectively. This result is consistent with the previous finding that complexes of much larger sizes and with higher aggregation rates occurred when the N/P ratio was larger than 2 [37]. The mean zeta potential of the DNA/PEI complexes (−22.1 ± 1.4, 16.7 ± 0.5, and 37.4 ± 0.9 mV) also increased with increasing N/P ratios (0.5, 3, and 7, respectively) (Fig. 1E & F).

The mean zeta potential of the cationic microbubbles was measured to be 52.7 ± 4.8 mV (Fig. 1G). The DNA/PEI complexes with an N/P ratio of 0.5 electrostatically attached to the cationic microbubbles with high efficiency, consequently decreasing the mean zeta potential of the microbubbles to − 3.8 ± 4.2 mV. In contrast, DNA/PEI complexes with an N/P ratio of 3 and 7 hardly attached to the cationic microbubbles, and modified the zeta potential of the microbubbles only slightly (49.67 ± 0.9 mV and 50.5 ± 0.8 mV, respectively). We also used confocal fluorescent microscopic imaging to examine the difference between cationic and neutral microbubbles with regard to loading the DNA/PEI complex (N/P: 0.5). As shown in Fig. 1H, no red fluorescence of propidium iodide (PI) dye (representing the existence of DNA/PEI complexes) was found on the neutral microbubbles, whereas the red fluorescence of DNA/PEI complexes was observed on cationic microbubbles, and the size of the region of red fluorescence increased with the size of the microbubbles. As shown in Fig. 1I, DNA/PEI complexes containing 250-ng DNA vaccine (N/P: 0.5) were incubated with different amounts of cationic microbubbles (2 × 10⁴ to 1 × 10⁷) for electrophoresis tests. The electrophoresis result showed that cationic microbubbles of 1 × 10⁶ could fully bind the 250-ng DNA vaccine (0.25 pg per microbubble) and avoid free DNA/PEI complexes moving towards the positive electrode.

3.2. In vitro transfection study of the DNA/PEI complex-loaded microbubbles

Using high-resolution confocal microscopy for imaging, we recorded the sonoporation and recovery process of the cell plasma membrane (labelled by the CellMask™ Orange, Fig. 2A) for cellular delivery. Microscale membrane disruption (indicated by white arrows, enlarged ROI, Fig. 2A) emerged at the location of the microbubble (indicated by white dashed circle) after ultrasound exposure (PD: 50 cycles; PNP: 0.58 MPa, single pulse) at 0 s, and the disruption gradually resealed within 300 s. We also recorded the transfer of DNA/PEI complexes from microbubbles to cells. As presented in Fig. 2B, without ultrasound exposure, the far-red fluorescence of DNA/PEI complexes (labelled by the Label IT™ Cy5) emerged at the position of the microbubble (indicated by a white arrow). With ultrasound exposure for inducing sonoporation, the microbubbles disappeared, and the fluorescence of DNA/PEI complexes emerged at the cellular areas (indicated by a yellow arrow). Of note, higher ultrasound pressure led to a larger amount of microbubbles that underwent collapse. All the microbubbles in the microscopic field of view collapsed and disappeared when ultrasound pressure increased to 0.58 MPa.

Fig. 2.

In vitro gene delivery and transfection experiments using DNA/PEI complex-loaded microbubbles. (A–B) Confocal microscopy imaging of the membrane perforation (A) and cellular delivery (B) characteristics of DNA/PEI complex-loaded microbubbles. (C) Representative images of the GFP expression results of different transfection groups. (D) Transfection efficiency of the four transfection methods shown in (C). (E) Transfection efficiency of DNA-loaded microbubbles using ultrasound excitations of different pressures. (F) Transfection efficiency of microbubbles loaded with DNA/PEI complex of different N/P ratios.

To study the cell transfection efficiency of different transfection methods, four experimental groups were used: 1) the DNA group, 2) the first sonoporation group with DNA-loaded microbubbles, 3) the DNA/PEI complex group, and 4) the second sonoporation group with DNA/PEI complex-loaded microbubbles. Representative microscopic images of the transfected cells expressing EGFP from each group with the highest transfection efficiency are displayed in Fig. 2C. The calculated cell transfection efficiencies of groups 1–4 were 0 %, 5.37 ± 1.88 %, 14.85 ± 2.76 %, and 27.78 ± 2.57 %, respectively (Fig. 2D). As for the DNA-loaded microbubbles, the cell transfection efficiency (0.13 ± 0.11 %, 0.76 ± 0.31 %, 1.49 ± 0.52 %, and 5.37 ± 1.88 %) increased with increasing ultrasound pressure (0.13, 0.28, 0.43, and 0.58, respectively) (Fig. 2E). When using an ultrasound pressure of 0.58 MPa for inducing sonoporation, all the microbubbles underwent collapse. the cell transfection efficiency (11.49 ± 0.98 %, 17.93 ± 2.53 %, and 27.28 ± 2.58 %) increased with increasing N/P ratios (0.5, 3, and 7, respectively) of the DNA/PEI complex-loaded microbubbles (Fig. 2F).

3.3. In vivo induction of intramuscular sonoporation

Using real-time ultrasound imaging, we examined the intramuscular injection of the microbubbles and the status of the microbubbles after ultrasound excitation. As depicted in Fig. 3A, the transfection medium (50 μL) containing DNA/PEI-loaded microbubbles (1.6 × 10⁶ microbubbles) was injected into the skeletal muscle of a mouse hind leg. Before the injection, no ultrasound echo signals were detected inside the murine muscle (Fig. 3B). During the injection, both signals representing the microbubbles and those representing the needle of the syringe (indicated by white arrows) could be seen in ultrasound images. After the injection, the transfection region inside the muscle could be identified. An ultrasound excitation pulse (PD: 50 cycles; PNP: 0.58 MPa, T: 2 min) for inducing sonoporation was then emitted. Different from their status in in vitro studies, the intramuscular microbubbles were not fully collapsed owing to the in vivo acoustic attenuation of the skin and part of the muscle tissue. Therefore, we increased the PD of the excitation ultrasound to 100 cycles to trigger the collapse of all the intramuscular microbubbles (Fig. 3C), which produced the highest transfection efficiency in cell transfection studies.

Fig. 3.

In vivo investigation of the sonoporation-mediated intramuscular transfection. (A) Graphical illustration depicting the intramuscular induction of sonoporation. (B–C) Ultrasound images showing the intramuscular collapse of microbubbles using ultrasound exposure pulse durations of 50 (B) or 100 (C) cycles. (D) Bioluminescence images of mice transfected with microbubbles of different types via ultrasound of different pressures. (E–F) Temporal profiles (E) and statistical results of the average bioluminescence radiance (F) of the mice shown in (D).

3.4. Effect of ultrasound pressure on gene expression over 400 days of observation

To examine the in vivo transfection efficiency of the proposed sonoporation methods, we employed a luciferase reporter gene for intramuscular transfection and conducted bioluminescence imaging for evaluating gene expression on days 1, 4, 7, 28, 190, 300, and 400 after sonoporation (Fig. 3D). Three experimental groups were set for investigating gene transfection (six transfection sites on six legs of three mice), including the control group, the first sonoporation group using DNA-loaded microbubbles, and the second sonoporation group using DNA/PEI complex-loaded microbubbles. In both sonoporation groups, 50 μL of transfection medium containing 10 μg of DNA and 1.6 × 10⁶ microbubbles was used for transfection.

For the first sonoporation group, we further included four subgroups of mice, which were transfected using ultrasound excitation with different pressures (0.13, 0.28, 0.43, and 0.58 MPa). On day 190, one of the three mice in the first sonoporation group (0.43 MPa) died owing to an obstruction of the airway during anesthesia. Temporal profiles of the luciferase gene expression are displayed in Fig. 3E; they indicate that the average radiance of bioluminescence peaked on day 4 and persisted for 400 days. A statistical analysis of the luciferase bioluminescence on day 4 revealed that the use of an ultrasound excitation with a higher amount of pressure resulted in more efficient gene transfection and expression (Fig. 3F). Furthermore, the newly-designed DNA/PEI complex-loaded microbubble (N/P ratio: 0.5) significantly elevated the transfection efficiency of the muscle. The DNA/PEI complex-loaded microbubbles induced a higher level (3.4-fold) of bioluminescence radiance ([1.1 ± 1.4] × 10⁵p/s/cm²/sr) compared with that induced by the DNA-loaded microbubbles ([3.3 ± 3.9] × 10⁴p/s/cm²/sr).

3.5. In vivo optimization of the N/P ratio of DNA/PEI complex

Three experimental groups were set to study the in vivo transfection efficiency of different transfection methods: the first group was injected with DNA only, the second group was injected with DNA/PEI complexes (N/P ratio: 0.5), and the third group was sonoporated with DNA/PEI complex-loaded microbubbles (N/P ratio: 0.5). In all the transfection groups, 50 μL of transfection medium containing 10 μg of DNA was used for intramuscular transfection and 1.6 × 10⁶ microbubbles were used for sonoporation. As shown in Fig. 4A and B, the luciferase expression of the third group ([1.1 ± 1.3] × 10⁵p/s/cm²/sr) was higher than those of the first and second groups (40.9-fold and 5.9-fold, respectively).

Fig. 4.

Optimization of the DNA/PEI complex N/P ratio for gene transfection. (A) Bioluminescence images of mice transfected via different methods. (B) Statistical results of the average bioluminescence radiance for the mice shown in (A). (C) Bioluminescence images of mice transfected with DNA/PEI complex-loaded microbubbles of different N/P ratios. (D) Statistical results of the average bioluminescence radiance for the mice shown in (C). (E) Masson staining images of the transfected muscle slices shown in (C). Scale bar: 100 μm.

Furthermore, we investigated the optimal N/P ratio of the DNA/PEI complexes loaded on the microbubbles for in vivo transfection, which had largely affected the in vitro cell transfection efficiency of the proposed sonoporation method. In all the sonoporation groups, 50 μL of transfection medium containing 20 μg of DNA and 1.6 × 10⁶ microbubbles was used for transfection. Compared with a DNA/PEI complex N/P ratio of 0.5, with increasing N/P ratios (NP ratios of 3 and 7), the transfection efficiency of the luciferase gene (eight transfection sites on eight legs of four mice) decreased 11.8-fold and 162.2-fold (p < 0.05), respectively (Fig. 4C and D). Histological analysis of the transfected muscles conducted using Masson staining revealed that the use of DNA/PEI complexes with increasing N/P ratios resulted in larger areas of muscle damage (indicated by black arrows, Fig. 4E).

3.6. Effect of DNA quantity and microbubble concentration on gene transfection

Different amounts of DNA/PEI complexes (N/P ratio: 0.5) carrying different quantities of DNA (1.6, 10, 20 and 100 μg) were mixed with 1.6 × 10⁶ cationic microbubbles for mediating intramuscular transfection. As shown in Fig. 5A and B, we found that the expression level of the luciferase gene of 20-μg was significantly higher than those of 10-ug (p < 0.05) and 1.6-μg (p < 0.01). As shown in Fig. 5C and D, the expression level of the luciferase gene of 100-μg was only slightly higher than that of 20-μg. We then held the DNA quantity (20 μg) of the DNA/PEI complexes fixed and added cationic microbubbles of different numbers (0.16 × 10⁶, 0.8 × 10⁶ and 1.6 × 10⁶) into the transfection medium for optimizing the microbubble parameter. As shown in Fig. 5E and F, it was found that microbubbles of 1.6 × 10⁶ significantly increased the expression level of the luciferase gene (p < 0.05). When the number of cationic microbubbles was further increased to 8 × 10⁶, the bioluminescence radiance of the expressed luciferase gene decreased even with a large quantity of DNA (100 μg) (Fig. 5G and H). Therefore, we suggest that optimal sonoporation parameters of 20-μg DNA and 1.6 × 10⁶ microbubbles should be used for subsequent intramuscular transfection experiment.

Fig. 5.

Optimization of DNA quantity and microbubble concentration for gene transfection. (A–B) Bioluminescence images of mice transfected with different quantity of DNA. (C–D) Statistical results of the average bioluminescence radiance shown in (A–B), respectively. (E–F) Bioluminescence images of mice transfected with microbubbles of different concentration. (G–H) Statistical results of the average bioluminescence radiance shown in (E–F), respectively.

3.7. Higher levels of gene expression induced by using split-dose and repeated transfection

In the development of a DNA vaccine against Zika virus, it was found that an in vivo transfection scheme using split-dose and repetition produced more efficient vaccination [8]. Therefore, we examined the effect of split-dose and repetition in sonoporation-mediated intramuscular gene transfection. As shown in Fig. 6A and B, when using sonoporation transfection medium with 20 μg of DNA and 1.6 × 10⁶ microbubbles, the split-dose approach with two and four injection sites (25 and 20 μL for each site, respectively) led to higher levels of luciferase gene expression ([7.2 ± 4.6] and [17.9 ± 4.6] × 10⁵p/s/cm²/sr (p < 0.05), respectively) when compared with the non-split-dose approach ([6.1 ± 2.9] × 10⁵p/s/cm²/sr). Furthermore, repetitive transfection of murine muscle on day 4 after the first transfection carried out on day 0 also significantly increased the level of luciferase gene expression (p < 0.05) (Fig. 6C and D). When the sonoporation-mediated intramuscular transfection protocol included both split-dosing (4 sites) and repetition (2 transfections), a further increase of 7.9-fold was observed in the bioluminescence radiance ([48.0 ± 18.4] × 10⁵p/s/cm²/sr) of the expressed luciferase (p < 0.0001).

Fig. 6.

Optimization of the sonoporation-mediated transfection schedule. (A) Bioluminescence images of mice transfected via 2 or 4 split doses. (B) Statistical results of the average bioluminescence radiance of the mice shown in (A). (C) Bioluminescence images of mice transfected via a schedule with or without repetition. (D) Statistical results of the average bioluminescence radiance of the mice shown in (C).

3.8. Immune responses induced by sonoporation-mediated DNA vaccination

After the transfection of a DNA vaccine, the induction of high-level immune responses requires efficient activation of the antigen-presenting cells [38]. Therefore, we examined, using immunofluorescence staining of the muscle slices, the migration and accumulation of the antigen-presenting cells (MHC Ⅱ positive) after sonoporation. As shown in Fig. 7A, the immunofluorescence of MHC Ⅱ (labelled by Alexa Fluor 647) was not obvious in the slice of non-treated muscle. In contrast, after the transfection of a HBsAg DNA vaccine using the optimized sonoporation protocol (ultrasound pressure: 0.58 MPa, N/P ratio: 0.5, DNA: 20 μg, microbubbles: 1.6 × 10⁶, 4 split-doses; a repetition on day 4), strong and specific immunofluorescence signals for MHC Ⅱ were found in some cytoplasmic areas of the muscle slice, indicating the accumulation of the antigen-presenting cells at the transfection site with the expression of HBsAg (labelled by green fluorescence) (Fig. 7B).

Fig. 7.

Immune responses evoked by sonoporation-mediated DNA vaccination. (A–B) Representative images of immunofluorescence staining for MHC-Ⅱ in slices muscle treated without (A) or with (B) sonoporation-mediated DNA vaccination. (C–D) Schematics of the time schedules for one-dose (C) and three-dose vaccinations (D). (E–F) The anti-HBsAg antibody levels measured after the vaccination using one-dose and three-dose schedules. (G–H) Comparison of antibody levels induced by injection-mediated (G) or sonoporation-mediated (H) vaccination using different dose schedules. (I) Representative microscope images of immunohistochemically stained slices of intact muscle or sonoporated muscle at different lengths of time after sonoporation.

To investigate the humoral immune responses of the optimized sonoporation vaccination protocol, we measured the anti-HBsAg antibody levels using ELISA and compared its immunization effects with those of DNA vaccination administered via injection. As shown in Fig. 7C and D, two vaccination schedules (one-dose and three-dose) were used for in vivo immunization, and HBsAg-specific antibodies were measured at the indicated time points. When using one-dose vaccination, the injection-mediated DNA vaccination failed to induce anti-HBsAg antibody production (Fig. 7E); in contrast, the sonoporation-mediated DNA vaccination significantly induced antibody production (p < 0.05) from week 4 (4.3 ng/mL), with the highest antibody level detected at week 48 (24.0 ng/mL). However, we also noticed that successful antibody induction did not occur in all of the vaccinated mice (3/5). For the three-dose DNA vaccination schedule, the anti-HBsAg antibody levels induced by the injection-mediated vaccination method significantly increased with the number of doses (p < 0.05) (Fig. 7F and G), whereas the rate of effective vaccination (3/5) for the sonoporation-mediated vaccination method was not improved (Fig. 7H). Compared with the injection-mediated vaccination group whose antibody levels peaked at week 12 and then gradually decreased, the sonoporation-mediated vaccination group had sustained antibody protection (56 and 52 weeks for one- and three-dose vaccination schedule, respectively).

3.9. Recovery of the sonoporated murine muscle

It has been reported that, during gene transfection, the negative effects of sonoporation induce some of the cells to undergo apoptosis [39]. Therefore, we examined the expression levels of a potent biomarker for apoptosis (caspase-3), using immunohistochemical staining, in muscle slices after sonoporation-mediated transfection. As show in Fig. 7I, caspase-3 was not expressed in the normal muscle tissue (enlarged ROI 1). In contrast, obvious expression of caspase-3 in the cytoplasm of muscle cells was visible (positive area < 4 %) by 1 day after sonoporation (enlarged ROI 2). The recovery of muscle cells after sonoporation-induced damage was apparent by 4 days after sonoporation (enlarged ROI 3). On day 7, the sonoporated muscle was fully recovered and showed a lack of immunohistochemical staining for caspase-3, similar to that observed in the control (enlarged ROI 4).

3.10. Safe DNA vaccination without chromosomal integration

To investigate whether sonoporation induced the genomic insertion of luciferase plasmids to enable stable luciferase expression for 400 days, we performed whole genome resequencing of murine muscles after transfection. Stable luciferase expression in murine legs was confirmed by bioluminescence imaging on day 7 and 35 after sonoporation-mediated transfection (Fig. 8A). Six muscle samples from three mice were excised on day 45 after transfection and mixed together to extract the total DNA. High-throughput DNA sequencing produced a total of 674.5 million reads (paired-end, average read length of 150 bp). Using Trimmomatic software (version 0.39), 604.3 million clean reads (mean insert size of 272.68 bp) were obtained. Using BWA software (version 0.7.17), 99.11 % of the clean reads were mapped to the reference genome (GenBank assembly accession: GCA_001632525.1). The mapped size of the murine genome is 2627327954 bp and its mean sequencing depth is 32.5X (Fig. 8B). The mapped size of the luciferase plasmid is 7070 bp and its mean sequencing depth is 46.24X (Fig. 8B).

Fig. 8.

Whole genome resequencing results of the sonoporation-transfected muscles. (A) Bioluminescence images of mice transfected with sonporation showing stable luciferase expression. (B) Mapping statistics of the sequenced murine genome and luciferase gene. (C) Circos plot showing chromosomes, GC content (GC: ratio of guanine and cytosine), sequencing depth, SNP count (SNP: single nucleotide polymorphism) and Indel count (displayed in order from outside to inside). (D) Plasmid map of the luciferase gene sequenced in murine muscle. (E) Comparison of the sequences of the luciferase genes extracted from E. coli and the murine muscle.

High-throughput DNA sequencing confirmed the existence of luciferase plasmids in the murine muscle cells. However, the insertion of luciferase plasmids in the murine genome was not detected when using three genome assembler software (SPAdes (version 3.14), NOVOPlasty (version 3.7) and MEGAHIT (version 1.2.9)). It was also confirmed that luciferase plasmids existed in the muscle cells in the closed circular form. Using the Genome Analysis Toolkit (GATK, version 4.1.1.0), we detected 497,494 single nucleotide polymorphism (SNP) variants as well as 282,990 insertion and deletion (Indel) variants (Fig. 8C). Using SnapGene Viewer software (version 7.0.1), common features of the sequenced luciferase plasmid was automatically annotated and plotted (Fig. 8D). DNA sequence comparison (similarity of 99.95 %) of the luciferase plasmids extracted from E. coli cultures and murine muscles were also visualized (Fig. 8E). Our results confirmed that sonoporation-mediated gene transfection is safe to avoid plasmid integration into the host genome.

4. Discussion

4.1. A new microbubble design for gene-loading and an optimized sonoporation protocol for transfection

As the ultrasound contrast agent for initiating sonoporation, microbubbles and their gene-loading ability play crucial roles in advancing sonoporation-mediated gene transfection. For example, microbubbles can be transported throughout the body with the circulating blood and the gene-loading ability of microbubbles enabled sonoporation to achieve targeted transfection in deep tissues (e.g., the liver and the heart) [19], [22]. It has also been demonstrated that sonoporation using cationic microbubbles loaded with genes can achieved higher transfection efficiency than that using neutral ones without gene-loading [40]. However, the previously-reported gene-loading ability of microbubbles is still relatively low (0.02–0.03 pg per microbubble) [35], [36]. In this work, we increased the gene-loading ability of the microbubble by providing a new microbubble design (Fig. 1). We first generated nanoscale DNA/PEI complexes and then electrostatically loaded the DNA/PEI complexes onto cationic microbubbles to attain an 8.3-fold increase in the DNA-loading capacity (0.25 pg per microbubble).

In in vitro studies using live-cell imaging and GFP gene transfection, our results revealed the membrane perforation and cellular delivery characteristics of the newly-designed microbubble under ultrasound excitation (Fig. 2). When using direct injection for in vivo luciferase gene transfection, DNA/PEI complexes produced a transfection efficiency 5.9-fold higher than that of the un-condensed DNA (Fig. 4A and B). One limitation of our study is that we used fibroblast cells instead of myoblast cells for in vitro cell transfection study. When the newly-designed microbubble with DNA/PEI complex loading was used for in vivo transfection, sonoporation further increased the transfection efficiency by 40.9-fold (Fig. 4A and B). Moreover, the sonoporation protocol for intramuscular transfection was optimized with respect to the following aspects: a) sonoporation parameters including peak negative pressure of the ultrasound, N/P ratio of the DNA/PEI complex, DNA quantity and microbubble concentration (Fig. 3, Fig. 4, Fig. 5); and b) sonoporation scheme including split-dose and repetition (Fig. 6). Using the optimized sonoporation protocol (ultrasound pressure: 0.58 MPa, N/P ratio: 0.5, DNA: 20 μg, microbubbles: 1.6 × 10⁶, 4 split-doses; a repetition on day 4), a further increase of 43.6-fold was observed in the intramuscular transfection efficiency.

4.2. Safe luciferase gene expression for 400 days and sustained antibody production for 56 weeks

Sonoporation has been considered capable of mediating only transient gene expression, thus limiting its application in therapeutic scenarios that require long-term transgene expression [13]. However, we recorded a long-term (400 days) intramuscular expression of sonoporation-transfected luciferase gene (Fig. 2D & E). In the literature, Lee et al. reported a prolonged intramuscular expression of sonoporation-mediated gene transfection of over 30 days [41]. Manta et al. reported a sustained sonoporation-mediated transgene expression in murine liver for 180 days [19]. It is reasonable to deduce that the long-term (400 days) gene expression reported in this study is due to the fact that skeletal muscle cells were terminally differentiated cells and the sonoporation-delivered luciferase plasmids underwent stable expression inside these cells. Notably, even when using the transfection method of intramuscular injection whose efficiency is relatively low, persistent luciferase gene expression for 19 months was observed [42]. When using electroporation for enhancing the intramuscular transfection, long-lasting expression of luciferase gene for 1 year was also observed [43]. Note that a small number of the plasmids transfected by electroporation were reported to randomly integrate into the genome of murine muscle cells [44]. Random genome integration in muscle cells has the risk of insertional mutagenesis, which is considered to be unsafe even in post-mitotic tissues [45].

To further examine the safety of sonoporation for transfection, whole genome resequencing of six muscle samples with stable luciferase expression was carried out. High-throughput DNA sequencing confirmed the existence and extrachromosomal state of luciferase plasmids in muscle cells (Fig. 8B-E). Therefore, the risk of chromosomal integration can be ruled out in sonoporation-mediated gene transfection. In this study, we found that microbubble collapse resulted in higher gene transfection efficiencies when compared with microbubble oscillation, which is consistent with the intracellular delivery results reported by Fan et al. [46]. Note that Suslick et al. reported that extraordinary conditions (e.g., temperatures up to 20,000 K and pressures of several thousand bar) occurred during microbubble collapse [47]. Although the reported extraordinary conditions could damage the integrity and quality of DNA plasmids, our high-throughput DNA sequencing results showed that the sonoporaion-delivered luciferase plasmids existed in the muscle cells in the intact circular form. Based on the microbubble collapse and shedding behavior recorded by ultra-high-speed cameras [48], [49], we suggest that the majority of the DNA/PEI complexes were shed from microbubble shells before the formation of local extraordinary conditions at the centers of the microbubbles.

Compared with intramuscular injection and electroporation which reported significant late decline in transgene expression [42], [43], sonoporation resulted in stable high-level expression of luciferase gene for 400 days (Fig. 2E). Note that long-term transgene expression is not necessary for DNA vaccination. Our results suggest that sonoporation-mediated gene transfection in terminally differentiated cells holds therapeutic potential for genetic diseases, such as Duchenne muscular dystrophy (DMD). Notably, the time point at which the sonoporation-transfected luciferase gene reached its peak level of expression varied among different sonoporation studies; Manta et al. and Lee et al. reported that the expression of luciferase gene peaked on day 2 and 8 after sonoporation, respectively [19], [41]; whereas here, we observed the maximum expression of luciferase gene on day 4 after sonoporation. Mechanistic insights are still needed to characterize the nuclear uptake dynamics of the sonoporation-delivered DNA plasmids.

Compared with intramuscular injection, the proposed sonoporation method effectively increased the magnitude of induced immune responses. First, evident accumulation of antigen-presenting immune cells (MHC Ⅱ-positive cells) was found in the sonoporation site of the murine muscle at 1 day after transfection (Fig. 7A & B). Second, when using a one-dose vaccination schedule, the mean serum anti-HBsAg antibody level of the sonoporation group was significantly higher than that of the injection group (p < 0.0001) (Fig. 7E). Lastly, long-lasting antibody production (56 and 52 weeks for one- and three-dose vaccination schedule, respectively) without obvious decline were detected in the sonoporation groups (Fig. 7E & F). However, successful antibody production was not observed in all the sonoporation-transfected mice (3/5). Although a three-dose vaccination schedule increased the antibody levels of the injection group, this schedule did not increase the antibody response rate in both the sonoporation and injection groups (Fig. 7G & H). Geissler et al. demonstrated that hepatitis B DNA vaccines only achieved antibody response rates of 20–80 % and the addition of complete Freund’s adjuvant increased the rates to 100 % [50]. Adjuvants, which increase the response magnitudes of the immune systems to antigens, are indispensable components of vaccines [51]. Therefore, we suggest the utilization of molecular adjuvants (e.g., DNA plasmids expressing cytokines and chemokines) together with sonoporation to further enhancing the immunogenicity of DNA vaccine.

Using ultrasound imaging, we found that the location and the range of the transfection region varied among different intramuscular injections (Fig. 3A). It has been reported that the distribution of DNA vaccine inside the murine muscle affected the immunogenicity of the DNA vaccine [52]. Therefore, in future work, a more consistent injection of the transfection medium can be achieved via the use of a fixed location and depth of the injection needle and monitored using real-time ultrasound imaging. In sonoporation-mediated transfection, the cavitation status of the microbubbles (e.g., stable oscillation or violent collapse) greatly affected the consistency of the transfection results [53]. Lo et al. developed a cavitation index (CI) parameter to regulate the total cavitation dose in a cellular transfection so that better control of the gene transfection consistency was achieved [54]. We also noticed that, in the research area of ultrasound and microbubble-mediated opening of the blood–brain barrier, closed-loop cavitation control has improved the consistency of intracranial drug delivery [55]. Therefore, real-time monitoring and dose control of microbubble cavitation can be implemented for in vivo transfection in future work to improve the consistency of sonoporation-mediated DNA vaccination.

5. Conclusion

Compared with mRNA vaccines, DNA vaccines are more stable in storage and less expensive in large-scale production. However, the practical application of DNA vaccines is still limited by their low transfection efficiency and immunogenicity. This study for the first time demonstrated the effectiveness and safety of sonoporation in improving DNA vaccination performance. By providing a new microbubble design with increased DNA-loading capacity and an optimized sonoporation protocol with high transfection efficiency, we promote technological development of sonoporaion as a promising non-viral transfection method. Using a hepatitis B DNA vaccine for immunization, we show that sonoporation effectively activated MHC-II⁺ immune cells and induced significantly higher serum antibody levels over a longer period (52–56 weeks) when compared with intramuscular injection. We also recorded the longest reported in vivo expression period (400 days) of a sonoporation-transfected luciferase gene. The first whole genome resequencing results of sonoporation-mediated transfection were presented in this study and confirmed that sonoporation achieved safe gene transfection without inducing chromosomal integration.

CRediT authorship contribution statement

Yuanchao Shi: Methodology, Investigation, Data analysis. Weixiong Weng: Writing – original draft, Investigation. Mengting Chen: Validation, Visualization. Haoqiang Huang: Validation, Methodology. Xin Chen: Conceptualization, Resources. Yin Peng: Resources. Yaxin Hu: Conceptualization, Supervision, Writing – review & editing.

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yaxin Hu reports financial support was provided by National Natural Science Foundation of China. Xin Chen reports financial support was provided by Shenzhen Basic Science Research.

Data availability

Data will be made available on request.