AIP Conference Proceedings 2155, 020033 (2019); https://doi.org/10.1063/1.5125537 2155, 020033
© 2019 Author(s).
The use of a needle-free injector for DNA vaccination in BALB/c mice
Cite as: AIP Conference Proceedings 2155, 020033 (2019); https://doi.org/10.1063/1.5125537 Published Online: 06 September 2019
Fera Ibrahim, Silvia T. Widyaningtyas, Ekawati Betty Pratiwi, et al.
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The Use of A Needle-Free Injector for DNA Vaccination in BALB/c Mice
Fera Ibrahim
1,2, a), Silvia T. Widyaningtyas
2,b), Ekawati Betty Pratiwi
2,c)and Budiman Bela
1,2,d)1Department of Clinical Microbiology, Faculty of Medicine, Universitas Indonesia - Dr. Cipto Mangunkusumo General Hospital, Jakarta, Indonesia.
2Virology and Cancer Pathobiology Research Centre, Faculty of Medicine, Universitas Indonesia - Dr Cipto Mangunkusumo General Hospital, Jakarta, Indonesia.
a)Corresponding author: [email protected]
Abstract. The capability of DNA vaccine to transfect cells determines the success of DNA vaccine, but there are some natural obstacles that reduce the efficiency of DNA delivery into cells. One way to overcome this problem is through the use of a delivery systems such as needle free injectors (NFIT). In the present study, we evaluated 2 kinds of NFITs, Injex and Twin-Jector EZII, and as control we use hypodermic syringes. The path of injection and the capability of NFITs to deliver DNA into cells were observed by using India ink and plasmid coding enhanced green fluorescent protein (pCDNA eGFP) respectively. A mass of 50 µg pcDNA3.1-eGFP in 50 µL of PBS and India ink were injected into the mouse thigh using these three delivery systems. Two days post-injection, histological slides were prepared from the thigh muscle and the expression of eGFP was observed by confocal microscopy. The results show that all the delivery systems used in the present study were able to deliver India ink and pcDNA3.1-eGFP to the thigh muscle. There was a difference in the spread of Indian ink produced 3 kinds of delivery systems used in this study. Furthermore, there was also a difference in the distribution of eGFP expression cells, that corelated with the pattern of the path /spread of fluid produced by the three delivery systems. The number and intensity cell expressing eGFP was intense in mice injecting with hypodermic syring compare to Injex and Twin-Jector EZII. - The results seen with India ink support the eGFP expression results. In conclusion, needle free injection can be used to deliver DNA into BALB/c muscle, but less efficient compared to hypodermic syringe.
INTRODUCTION
DNA vaccines have been developed with a view to control numerous diseases including viral and bacterial infections and certain cancers [1-3]. The effectiveness of DNA vaccines depends on the type of cells that are transfected to produce the expected antigen [4]. Antigen expression relies on the protein biosynthesis machinery of host cells to recognize not only the transcription and translation unit but also the triplet codon of the DNA in the vaccine [5-6]. Under certain conditions, the host-biosynthesis machinery fails to assist the expression of the antigen encoded by the DNA vaccine due to codon bias, during which there is a discrepancy in the distribution of codons for similar amino acids between the host and the DNA vaccine [7-9].
Trials of DNA vaccines in small animal models, predominantly mice, have produced a promising immunogenic response; however, this response can rarely be replicated in larger animals [4]. The main obstacle of DNA vaccine usage in large animals, non-human primates, and humans is the need for high doses to induce an immune response [10]. To avoid this obstacle, certain delivery systems have been developed, one of which is needle-free injection technology (NFIT) [11-13]. First developed in 1940 to facilitate the mass vaccination process, NFIT has
some advantages such as avoidance of contamination, use in people with a phobia of needles, and a reduction in pain [14].
NFIT technology utilizes pressure to force the liquid or powder to penetrate the skin without interfering with its integrity [13,14]. NFIT consists of components to generate high pressure, a drug reservoir, and a nozzle [11,15- 16]. Based on the source of pressure, there are two types of NFIT: spring-loaded and gas-powered jet injectors [17].
The spring-loaded jet injector model works by means of a spring mechanism; the spring is drawn back, and when the trigger is activated, the spring is released to create high pressure that injects the drug into the skin [17]. In the gas- powered model, a cylinder containing high-pressure gas is connected to the NFIT, when the trigger is activated, the pressure from the gas cylinder drives the drug to penetrate the skin [17]. Another model for generating pressure is the battery-powered jet injector [11]. The NFIT compartment where high pressure forms is known as the piston [15]; to increase drug penetration into the skin, Li et al., (2016) added short needles of ≤ 5 mm in length [12]. Another part of the NFIT that contributes to the control of drug penetration is the nozzle, which is connected to the drug reservoir by the skin [11], and its orifice diameter controls the diameter and speed of the drug stream [16]. The final part of the NFIT is a scaled drug reservoir that is available in various volumes [16].
To evaluate the capability of DNA delivery system such as NFIT in delivering liquid, markers such as India ink is used. India ink is a carbon particle with bacterial size that can be used to track the distribution of particles, cells or tissue in living system since decades [18]. Ren et al (2002) using India ink to measure the length of the injection path of India ink that injected using jet injection to animal model [19-20]. It is strongly recommended to try the injection system in animal model by using colored liquid such as India Ink prior injecting DNA vaccine [21]. GFP is molecule that can absorb and emit light at different wavelength and widely used as reporter protein. To increase its intensity and stability GFP has been modified [22]. The new variant of GFP later called as enhanced Green fluorescent protein (eGFP) [23]. Now days, eGFP have been used widely in studying biological processes in numerous species such as bacteria, fungi, plant, insect and worm [24]. In DNA vaccine study, plasmid coding the eGFP protein was used to measure the efficiency of DNA transfection and the distribution of vaccine in body [25-26].
Essentially, NFIT is developed to improve the efficiency of intradermal drug/vaccine delivery to certain human body parts [17]; however, some researchers have used NFIT, such as ShimaJet and PoederJet, to evaluate the immunogenicity of DNA vaccines in mice [27-28]. Accordingly, in the present study, we analyzed the performance of NFIT, Injex, and Twin-Jector EZ II in the delivery of India ink and DNA encoding enhanced green fluorescent protein (eGFP) to mouse thigh muscle, followed by evaluation of the immunogenicity of DNA vaccines in BALB/c mice.
MATERIALS AND METHODS pcDNA3.1-eGFP
The pcDNA3.1-eGFP plasmid uses the pcDNA3.1 (Invitrogen) backbone as a vector and eGFP as a coding gene that has been optimized for the mammalian system. pcDNA3.1 contains a transcription and translation unit for mammalian cells, which includes an enhancer, a cytomegalovirus (CMV) promoter, a ribosomal binding site, and a bovine growth hormone (BGH) polyadenylation signal. The plasmid was propagated in Top10 Escherichia coli cells in LB liquid medium (HiMedia) containing ampicillin (100 μg/mL). Plasmid DNA was obtained as described in the Qiagen Plasmid Mega Kit and confirmed by enzyme restriction analysis and Sanger sequencing.
Animals
BALB/c mice, aged 6−8 weeks, were obtained from PT Biofarma and maintained under standard housing conditions. All experiments were approved and conducted in accordance with the Animal Care SOP of the Virology and Cancer Pathobiology Research Centre. All experiments were approved by the ethical committee of Medical Faculty of Universitas Indonesia (No. 405/II2.F1/ETIK/2014) and performed following the guidelines of ethical committee of Medical Faculty of Universitas Indonesia.
Delivery Systems
Injex, produced by Rösch AG Medizinetechnik, is a liquid-based needle-free injection system. The ampoule volume is 3 mL and the orifice diameter is 0.17 mm. Twin-Jector EZII (Antares, marketed by JCR Pharmaceutical Co. Ltd, for human growth hormone (hGH) in Japan) is also a liquid-based needle-free injection system. The diameter of the orifice is approximately half gauge 30 (+0.128 mm). A 1-mL disposable hypodermic needle was used as the control delivery system (Figure 1).
FIGURE 1. Delivery systems used in the present study. (a) Hypodermic syringe; (b) Injex; (c) Twin- Jector EZII; (d) drug reservoir (d1 Injex; d2 Twin-Jector EZ II).
Fluid Stream
The fluid stream generated by the hypodermic syringe, Injex, and Twin-Jector EZII, was observed using India ink diluted 1:7 in PBS as a marker. Prior to injection with India ink, BALB/c mice were terminated by cervical dislocation.
A volume of 50 µL India ink was injected into the mouse thigh muscle. The methods of injection can be found in Figure 2. Injection using a hypodermic syringe was performed into tight muscle. Prior to injection using NFIT, the femur was bent caudally towards the mouse (Figure 2(b)) and the NFI was placed in the ventrocranial direction (Figure 2(c) and (d)).
FIGURE 2. Injection method. (a) Hypodermic syringe; (b) femur bent caudally towards the mouse; (c) Injex; (d) Twin-Jector EZ II.
Following injection, a blunt section was performed to separate the skin from the thigh muscle. The distribution of India ink, the presence of lesions, and other occurrences were reported.
Expression of Enhanced Green Fluorescent Protein
The capability of Injex, Twin-Jector EZ II, and a hypodermic syringe to deliver pcDNA3.1-eGFP was determined by the expression of eGFP in mouse muscle tissue. Prior to injection with 50 µg plasmid diluted in 50 µL sterile PBS, mice were anesthetized using xylazine-ketamine. The injection methods were performed as described above. Two days after injection, the animals were terminated by cervical dislocation. Histological slides were prepared from the mouse thigh muscle, and expression of eGFP was observed using a confocal laser scanning microscope (Fluoview FV1000; Olympus). eGFP was excited at 488 nm, and emission was detected using a standard fluorescence filter set (522 + 35).
RESULTS
The stream of India ink in the thigh muscle following three kinds of injection methods can be observed in Figure 3. Following injection using a hypodermic syringe, the India ink was predominantly distributed surrounding the muscle injection site, with only a small amount found at the other side of the thigh. Moreover, the distribution of India ink on the skin or subcutaneously could not be observed (Figure 3 (a) and (b)). Following injection using Injex, the India ink was spread evenly throughout the thigh muscle, not only at the site of injection but also at the other side of the thigh (Figure 3(c) and (d)). However, we observed the presence of perforation at the site of injection, with the India ink spreading out to the environment (Figures 3(d) and 4). Following injection using the Twin-Jector II, the stream of India ink was more concentrated in the skin area than in the muscle (Figure 3(e) and (f)).
FIGURE 3. The India ink stream in muscle and skin. Injection using a hypodermic syringe (a−b). (a) Distribution of India ink at the site of injection using a hypodermic syringe; (b) distribution of India ink at the opposite side of the thigh. Injection using Injex
(c−d). (c) Distribution of India ink surrounding the thigh muscle; (d) penetration of India ink to the opposite side of the thigh (red arrow shows the India ink that passed through the thigh muscle). (g−i) Injection using Twin-Jector EZ II. (g) Site of penetration;
(h) distribution of India ink in the subdermis (black circle); (i) distribution of India ink in the muscle (yellow arrow).
FIGURE 4. Skin perforation following injection using Inject.
Injex, Twin-Jector EZII, and a hypodermic syringe were able to deliver DNA into the thigh muscle. Delivered DNA passively transfected thigh muscle cells as shown by the expression of eGFP. Moreover, the expression of eGFP shows the ability of the biosynthesis machinery of muscle cells to recognize the transcription and translation unit in the pcDNA3.1-eGFP backbone. Qualitatively, the expression of eGFP was found most frequently in the muscle cells injected using the hypodermic syringe as compared with that injected using Injex and Twin-Jector EZII (Figure 3).
The expression of eGFP in muscle injected with a hypodermic syringe was concentrated in a certain location; however, the expression of eGFP in muscle injected using Injex and Twin-Jector EZ II was diffuse (Figure 3).
Figure 5. Green fluorescent protein expression in mouse thigh muscle. (a) Control thigh muscle from mice not injected with pcDNA3.1-eGFP; (b) thigh muscle injected with pcDNA3.1-eGFP using a hypodermic syringe; (c) thigh muscle injected with
pcDNA3.1-eGFP using Injex; (d) thigh muscle injected with pcDNA3.1-eGFP using Twin-Jector EZII. A FITC filter was used to capture emission following excitation of eGFP at 488 nm. TD1: transmission/bright field image; magnification: 20x objective.
DISCUSSIONS
The effectiveness of DNA vaccination in inducing immune responses was initially tested by injecting naked plasmid intramuscularly in a murine model to produce antigens in passively transfected muscle cells [29]. In this method, triggering an immune response relies on DNA vaccine antigens to induce inflammation that attracts leukocytes [30]. The present study showed that injecting DNA using a hypodermic syringe was more efficient in delivering DNA as compared with using Injex and Twin-Jector EZ II. The efficiency of a hypodermic syringe in delivering naked DNA has also been reported by Wolf and Budker [31]. The difference in the number of transfected cells following injection using the three different delivery systems was likely related to the fact that the spread of DNA in the mouse thigh muscle and surrounding tissue was unique for each tool.
Injection of India ink using a hypodermic syringe has showed that the distribution of India ink was localized at the injection site. When using Injex, the India ink spread evenly throughout the thigh muscle; the fluid injected with high pressure was spread in all directions and followed a route that lacks resistance, especially the perimysium or epimysium [32]. Furthermore, high pressure injection can cause muscle fibres to be torn and severed [32]. Injex hascaused perforation in mouse skin and penetration of the fluid to other side of the thigh muscle. The loss of a small amount of liquid can significantly affect the drug/vaccine dose.
In NFIT, the depth of drug/vaccine penetration depends on several factors such as the speed at the nozzle exit, drug/vaccine mass, and injection duration [33]. The NFIT produced a constant high pressure of approximately 1420−1800 psi (pounds per square inch), helping the drug/vaccine to penetrate the skin and subcutaneous fat without damaging the integrity of either [13,34]. Due to differences in the thickness of the skin at different body parts, NFIT does not work universally [17]. The current commercialized NFIT was optimized to deliver drug/vaccines to humans [32,34] The differences in the skin thickness between humans/large animals and small animals need to be considered when using NFIT as a delivery system; however, a special injection technique, ShimaJET, which prevents liquid leakage, was used to determine the effectiveness of a DNA vaccine against Influenza [28].
In some cases, the high pressure produced by NFIT induces contamination. When liquid or solid material hits the skin at high velocity, there is a possibility that the material will splash back and contaminate the nozzle [35].
Blood in the nozzle could be sucked back and contaminate the next dose [36]. The usage of high pressure under certain conditions can create a hole in the skin, and when the stream of drug mixes with the tissue, fluid and blood can move back into the hole, against the incoming drug, and back into the nozzle [11]. In the present study, we observed that NFIT could penetrate through the mouse thigh, which must be seriously considered, since it not only endangers operator health but could also contaminate the environment.
CONCLUSION
Needle free injection can be used to deliver DNA into BALB/c muscle, but less efficient compared to hypodermic syringe.
ACKNOWLEDGMENTS
This research was funded by the Ministry of Research, Technology, and Higher Education of the Republic of Indonesia through the Insinas program.
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