A novel use of a needle-free injection system for improved nucleic acid delivery and expression in vivo

Transfection refers to nonviral methods of nucleic acid delivery for gene expression or silencing utilizing physical or chemical methods [1–3]. Transfection has been used extensively in various in vitro experiments [4] and more recently adapted for in vivo applications, including the treatment of genetic and nongenetic clinical conditions [5–8]. Some of these procedures have been approved by the US FDA and the EMA [6].

Despite the recent advancements in transfection methodologies, there are still underlying challenges to achieving high-efficiency gene delivery in vivo [7]. Electroporation, an effective physical transfection method that uses localized electrical voltage to transiently increase cell membrane permeability for genetic materials is associated with cell death, increased costs and potential surgical contamination risks [1,2]. On the other hand, chemical transfection, which utilizes cationic polymers/peptides forming complexes with the negatively charged nucleic acids, is less efficient compared with electroporation [3].

Conventional needle-based in vivo delivery of nucleic acid and transfection reagent mixtures is widely used and considered the most convenient method. However, this method may not yield high transfection efficiency. In addition, comparative performance analyses of the needle-based and needle-free delivery systems are not available. Recently, needle-free injection devices for human applications in dermatology, vaccination, podiatry and veterinary practices have been developed [8]. The MESO-JET (MJ) and the MED-JET H4 MULTIJET (MJH4M) (Figure 1A) are different models of noninvasive, low-pressure, needle-free injection systems manufactured by Medical International Technologies (MIT Canada) Inc. (QC, Canada). Both devices utilize compressed carbon dioxide (CO₂) to deliver a low-pressure, ultra-fine jet stream that penetrates the skin to allow for intradermal, subcutaneous, and intramuscular injections by adjustment of the injection pressure [8]. Both systems use a power supply, a CO₂ cylinder, a pressure regulator, and a handpiece. The CO₂ cylinder is connected to a pressure regulator that controls the pressure per square inch (psi) transmitted into the handpiece, allowing delivery of volumes ranging from 50 to 300 μl. Both pressure and volume are controlled by the operator (Figure 1A).

The handpieces, the delivery process and the power supply between the two systems are different. The MJ handpiece includes two female inlets: one at a 30° angle in the center of the handpiece for connecting the syringe with the injectable product, and a 5-mm disposable plastic tip through which the delivery material is administered. Alternatively, the MJH4M handpiece, shaped as an ergonomic gun, differs in that it incorporates a disposable sterile cartridge to be filled by the user and inserted into the barrel of the handpiece. The barrel also serves as a dose-adjuster knob. In addition, the MJH4M device has a power adjustment knob that controls the delivery of each injection by regulating the power. The pressure is controlled by the adjustment of the regulator. For the MJ system, pressing the foot pedal activates the delivery of the product through a plastic tip, whereas for the MJH4M system this occurs by pressing the trigger, activating the delivery of the material through a disposable sterile cartridge. The MJ device is composed of a separate power box and a power unit, whereas the MJH4M has the power unit within the ergonomic handpiece.

The improved MJH4M device was developed to deliver multiple volumes using the same device from 25 to 300 μl in increments of 25 μl compared with the MJ device with its fixed volume of 50 μl only. The initial proof-of-principle experiments were performed using the MJ device. Later, when the MJH4M device became available, it was used as the primary device. Before performing the in vivo experiments, we compared the dispersion of 1% toluidine blue solution in ballistic gel solution upon delivery by a needle or the MJH4M system. As shown in Figure 1B, dye delivered via the MJH4M system was well dispersed, while no dispersion was observed for the needle delivery.

The in vivo experiments were performed at the animal facility at Shriners Hospital for Children (QC, Canada) following an approved animal user protocol. Mice were kept in a 12-h day/night cycle and fed laboratory chow and water ad libitum. First, 1% toluidine blue solution was delivered using the MJ system into the hindlimb muscles below the knee of adult C57BL/6 mice. For this, mice were anesthetized via a mixture of inhaled isoflurane and oxygen, and the ventral regions of the hindlimbs were shaved to expose the skin. The tip of the device handpiece was placed on the skin ventrally below the knee at the 7-mm mark (Figure 1C). The pedal was then pressed, and the device held on the hindlimb for 3 s to ensure the retention of the injected volume.

At 70 psi and 50 μl volume, the distribution of 1% toluidine blue was limited to the skin. Maintaining the pressure at 70 psi and increasing the volume to 100 μl led to a partial distribution of the dye in the superficial area of the muscle, while increasing the pressure to 80 psi led to a complete distribution of the dye in the hindlimb muscles (Figure 1C).

Using the above optimized parameters, the in vivo transfection efficiency of the MJ device was then examined via injections of a Renilla luciferase plasmid (pR-Luc) DNA mixed with different delivery solutions such as calcium phosphate (CaP), K2^® Transfection Reagent (Biontex, Munich, Germany) or 5% glucose with 5% glucose without DNA as the negative control (NC). In all experiments, a pR-Luc plasmid concentration of 0.1 μg/μl was used. Mice were euthanized 48 h after the injection using inhaled isoflurane and carbon dioxide followed by cervical dislocation. The skin was removed from the hindlimbs and muscles below the knee were dissected out and flash frozen in liquid nitrogen. Next, the extracted tissues were homogenized in passive lysis buffer (Promega, WN, USA). Transfection efficiency was examined using a Dual-Luciferase^® reporter assay (Promega). The reporter gene activity was measured in relative light units per second per milligram (RLU/s/mg) of protein using a luminometer (Zylux, TN, USA). All measurements were made in duplicates. A Bicinchoninic Acid Protein Assay (Thermo Fisher Scientific, MA, USA) was used to determine protein concentration, which was used for normalization. Figure 2A shows the experimental steps.

GraphPad Prism 9 software (CA, USA) was used for all statistical analyses. Analysis of variance (ANOVA) and post-hoc Dunnett's and Tukey's multiple comparisons were used. Statistical significance was set at p < 0.05. As presented in Figure 2B, there was no difference between the relative luciferase activities in the muscle extracts from the NC (5% glucose-only) group and those from the group that received pR-Luc plasmid in CaP solution. The extracts from the group that received pR-Luc plasmid in the K2^® transfection reagent showed higher luciferase activity, but it was not significantly different from the NC group. A significantly increased luciferase activity (p = 0.006) was detected only in the group that received the pR-Luc plasmid in 5% glucose when compared with the NC (Figure 2B).

The newer MJH4M device was easier to use with similar delivery efficiency as shown by dye delivery experiments (Figure 3). However, while using this device we noticed leakage of fluid outside the leg muscles. This issue was resolved by reducing the delivery volume to 75 μl, injected at 80 psi as before. The transfection efficiencies for the pR-Luc plasmid delivery in saline, milli-Q water, or 5%, 10%, 15% or 20% glucose solutions were examined. The transfection reagents TurboFect (Thermo Fisher Scientific) and DNA-fect (RJH Bioscience, AB, Canada) were also evaluated. TurboFect is a lipid-based transfection reagent, while DNA-fect is a synthetic amphiphilic polymer-based transfection reagent. In addition, transfection efficiency of the MJH4M device was also compared to that of the needle injections.

Using the MJH4M device, the transfection efficiency for the pR-Luc plasmid using 5% glucose as the delivery vehicle was significantly higher (p = 0.022) than that of the nontransfected negative controls (NC) and showed the tendency to outperform but was not significantly higher than when saline (p = 0.195) or mqH₂O (p = 0.202) was used as delivery vehicles (Figure 4A). Next, the same amount of pR-Luc plasmid was delivered in 5%, 10%, 15% or 20% glucose; the 5% glucose–DNA combination appeared to result in the best transfection efficiency (p = 0.010 compared with 20% glucose-DNA combination) (Figure 4B).

For needle deliveries, the needle was inserted ventrally halfway into the hindlimb muscles below the knee. In comparison to the needle injections, the needle-free MJH4M device demonstrated superior transfection efficiency (Figure 5). Using 5% glucose as the delivery vehicle for the pR-Luc plasmid, we observed a 14.6-fold increase (p < 0.001) in luciferase activity when using the MJH4M device compared with the conventional needle injections (Figure 5). Delivering pR-Luc using a commercial in vivo DNA delivery reagent, TurboFect, produced a 16.2-fold increase (p = 0.016) in luciferase activity when compared to the conventional needle injections (Figure 5). Finally, another commercial transfection reagent, DNA-Fect, also showed a mild but significantly increased (p = 0.037) luciferase activity between the needle-based and needle-free deliveries of the pR-Luc plasmid. However, the DNA-Fect formulation delivered via the MJH4M device showed a lower transfection efficiency averaging 8248 RLU/s/mg protein compared with TurboFect or 5% glucose (299,963 RLU/s/mg protein and 367,438 RLU/s/mg protein, respectively).

Conclusion

We established a protocol for in vivo DNA transfection using the novel MJ/MJH4M needle-free injection systems developed by MIT Canada. The results demonstrated that the needle-free devices achieve superior transfection efficiency compared with DNA delivery via a needle injection. The needle-free technique holds potential for improved in vivo gene delivery that can be used in clinical therapy, including the treatment of genetic diseases and DNA vaccine delivery. Furthermore, by eliminating the use of needles, a common source of fear and discomfort, this effective and efficient needle-free delivery system can significantly enhance patient experience and overall acceptance. Future research should focus on the optimization of the delivery conditions, particularly using improved formulations for delivery agents, and performing comparative studies with established techniques such as electroporation to further validate the efficacy and the potential use of the needle-free approach.

The protocol can be found at protocols.io [9] and in the Supplementary Files.