In the biomedical field, researchers are actively investigating the ability of ultrasound to enhance efficacy of a variety of pharmaceutical molecules including cancer drugs [14–16]. A particularly promising cancer drug is antisense oligonucleotides (ASOs). ASOs are short sequences of nucleotides that could inhibit a specific messenger ribonucleic acid (mRNA) in cells. It has great potential because it enables rational drug design based on genomic information [17]. However, like many other hydrophilic molecules, one of the major challenges of using ASOs is the inefficient cellular uptake, which limits their therapeutic efficacy [14].
The most efficient tools to introduce nucleic acids such as deoxyribonucleic acid (DNA) and mRNA into cells are viruses. However, concerns for safety limit the use of viral-mediated transfection. A widely used nonviral method to enhance transfection is the addition of cationic liposomes [18,19]. More recently, ultrasound was found to further enhance the transfection of DNA that are complexed with cationic liposomes [20–24]. Koch et al. reported that ultrasound enhancement of liposome-mediated cell trans-fection is caused by cavitational effects [23]. However, the precise mechanism involved remains unclear.
Transfection could possibly be enhanced by sonoporation—the formation of transient pores on cell membranes due to cavitations. Cavitation might also disrupt the endosomes containing the uptaked nucleic acids and allow the nucleic acids to evade lysosomal degradation.
Despite the exciting possibility of using ultrasound to enhance the efficacy of ASOs, researchers have only been using conventional ultrasonic transducers. Treatment with conventional ultrasonic transducers would require multiple clinical visits for the ASOs injections as well as ultrasound expo-sures. Unwanted tissue heating and skin lesions due to the extracorporeal application of ultrasound also can present problems. Instead, an implantable MUT is proposed with an integrated controlled-release mechanism. Figure 5.4 shows a schematic diagram of the proposed device. Such a device could
FIGURE 5.4 Proposed device for enhancing efficacy of cancer drugs including ASOs (not to scale). The operating sequence is marked in numerical order.
Cells Ultrasonic pressure
wave front
(2) Cell wall temporary opens when bubbles collapse
(3) DNA pushed in by pressure waves
(1) Cavitation bubbles form
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be implanted into/near a tumor with minimally invasive surgery. It could then automatically release the ASOs and transmit ultrasound over time. Therefore, it could reduce treatment cost as well as patient discomfort.
A series of preliminary experiments have been carried out in our laboratory to investigate the possible use of MUTs to increase the efficacy of ASOs. The ASOs used in the experiments target the bcl-xL mRNA, which has been investigated previously for cancer therapy [25,26]. The cell lines investigated by us were (1) human umbilical vein endothelial cells (HUVEC) and (2) human prostate cancer cells (PC3). In our experiments, three different controls were used. The first was the untreated wells, which served as a negative control and gave the number of viable cells without any treatment.
The second control gave the number of viable cells after sonication. In the third control, Lipofectin, a state-of-the-art cationic lipid transfection agent, was added in conjunction with the ASOs but the wells were not sonicated.
For the HUVEC, a fair amount of cells were lysed due to sonication alone. After transfection of ASOs using lipofectin, only about 72% of cells remained viable. Sonication has enhanced the efficacy of ASOs+lipofectin and decreased surviving cells to 62%. The result of the experiment is summarized in Figure 5.5. Student’s t-test has shown that the result from ASOs+lipofectin+sonication is significantly different (p < 0.01) from sonication and ASOs+lipofectin.
For the PC3, sonication and ASOs+lipofectin individually did not contribute to cell death. However, ASOs+lipofectin+sonication enhanced the ASOs efficacy and only 93% of the cells survived. Student’s t-test again showed that the result from ASOs+lipofectin+sonication were significantly different (p <
0.01) from sonication and ASOs+lipofectin. Figure 5.6 illustrates the PC3 result.
Our experiments have shown that sonication with the prototype ultrasonic transducer enhances bcl-xL ASOs efficacy in HUVEC and PC3 cells. However, there existed a large number of parameters in our experiments, and they have not been systematically optimized. These parameters include reagent con-centration, sonication frequency/power/duration, incubation time, and so on. In the future, we will systematically investigate the effect of each parameter on the results. In this manner we will be able to optimize the synergetic effect of ultrasound and possibly further enhance the ASOs efficacy. We are also continuing our work on the fabrication of miniaturized prototypes and the MUT. Once those devices are fabricated, we plan to use them for repeating the experiments.
FIGURE 5.5 HUVEC viability after ASOs transfection in different conditions. ASOs+lipofectin+sonication resulted in a significant (p < 0.01) decrease of surviving cells compared to sonication and ASOs+lipofectin.
100%
Error bars show +/–1 standard deviation HUVEC Viability After ASO Transfection
80%
60%
Percentage cell survival
40%
20%
0%
Sonication (n = 3)
ASO + Lipofectin (n = 3) Conditions
ASO + Lipofectin + Sonication (n = 3)
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FIGURE 5.6 PC3 viability after ASOs transfection in different conditions. ASOs+lipofectin+sonication resulted in a significant (p < 0.01) decrease of surviving cells compared to sonication and ASOs+lipofectin.
120%
100%
Error bars show +/–1 standard deviation PC3 Viability After ASO Transfection
80%
60%
Percentage cell survival
40%
20%
0%
Sonication (n = 4)
ASO + Lipofectin (n = 6) Conditions
ASO + Lipofectin + Sonication (n = 9)
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