The Application of Clinical Lithotripter Shock Waves to RNA Nucleotide Delivery to Cells

AbstractThe delivery of genes into cells through the transfer of ribonucleic acids (RNAs) has been found to cause a change in the level of target protein expression. RNA-based transfection is conceptually more efficient than commonly delivered plasmid DNA because it does not require division or damage of the nuclear envelope, thereby increasing the chances of the cell remaining viable. Shock waves (SWs) have been found to induce cellular uptake by transiently altering the permeability of the plasma membrane, thereby overcoming a critical step in gene therapy. However, accompanying SW bio-effects include dose-dependent irreversible cell injury and cytotoxicity. Here, the effect of SWs generated by a clinical lithotripter on the viability and permeabilisation of three different cell lines in vitro was investigated. Comparison of RNA stability before and after SW exposure revealed no statistically significant difference. Optimal SW exposure parameters were identified to minimise cell death and maximise permeabilisation, and applied to enhanced green fluorescent protein (eGFP) messenger RNA (mRNA) or anti-eGFP small interfering RNA delivery. As a result, eGFP mRNA expression levels increased up to 52-fold in CT26 cells, whereas a 2-fold decrease in GFP expression was achieved after anti-eGFP small interfering RNA delivery to MCF-7/GFP cells. These results indicate that SW parameters can be employed to achieve effective nucleotide delivery, laying the foundation for non-invasive and high-tolerability RNA-based gene therapy

Lithotripter shock wave induced RNA-based gene therapy

Gene therapy is the process of introducing genes to augment or minimise the expression of absent or defective genes, respectively. Non-viral gene delivery systems for the treatment of genetic diseases, have evolved into highly appreciable nucleic acid-based therapies due to considerably less risk of host immunogenicity and induction of inflammatory responses. However, they are challenged by limited delivery. Thus, more efficient strategies are continually being sought. Lithotripter shock waves (LSW) are powerful acoustic waves that are an attractive choice of delivery system, as they offer a non-invasive, targeted and safe approach. Furthermore, the delivery of messenger ribonucleic acid (mRNA) possesses several advantages over commonly delivered plasmid deoxyribonucleic acids (pDNA), because it does not require opening of the nuclear envelope, thereby reducing the level of cell injury necessary for transfection. This work presents the first investigation on the efficacy of LSW mediated mRNA delivery, based on optimised SW parameters that balance the desired enhanced permeability of cell membranes against undesired cytotoxicity, and maintain the structural and biological stability of the RNA. A transfectability measure that defines the ability of SWs to permeabilise a cell whilst keeping it alive was established for dissimilar cell types, as a function of the acoustic pressure and number of SWs. Statistically significant RNA uptake was recorded in a tissue mimicking system, and using RNA analogues at various concentrations, the SW induced bio-distribution was characterised. In addition to LSW induced gene augmentation using mRNA, it was shown that LSWs could be used to effect gene inhibition through the delivery of siRNA. Kinetic experiments were carried out to measure mRNA uptake during shock wave exposure and indicated that rate of delivery was highest at the start of the SW dose and decreased during treatment. The results also suggested that the enhancement of cell permeability was significantly transient, and that mRNA was highly susceptible to degradation in its naked state. Furthermore, mRNA-based gene expression was shown to be predictive but quantal. The in vitro tissue model was improved from a gel-based system, to one that incorporated multi-cellular spheroids which capture aspects of 3-D tumours. Static overpressure was applied during SW exposure in order to suppress cavitation effects and isolate effects that could be attributed to shear due to cell-to-cell coupling. The results showed that mild overpressure improved RNA uptake the most, but that at higher overpressure, the level of increase in RNA uptake relative to controls, was dependent on the type of RNA nucleotide being delivered. This suggested that a complex interaction between LSW cavitation and direct stress dominates delivery. A final report was on the significant improvement of gene delivery when mRNA was encapsulated within a lipid nanoparticle vector, and SW exposure was assisted by cavitation agents. Also, by exposure to another acoustic stimulus – focused ultrasound (US), direct comparisons were made between SWs and US on the efficiency of delivery and tissue penetration. In conclusion, this thesis has shown that by choosing parameters appropriately, shock waves can be a promising strategy for the delivery of genes to cells.</p

Lithotripter shock wave induced RNA-based gene therapy

Gene therapy is the process of introducing genes to augment or minimise the expression of absent or defective genes, respectively. Non-viral gene delivery systems for the treatment of genetic diseases, have evolved into highly appreciable nucleic acid-based therapies due to considerably less risk of host immunogenicity and induction of inï¬ammatory responses. However, they are challenged by limited delivery. Thus, more eï¬cient strategies are continually being sought. Lithotripter shock waves (LSW) are powerful acoustic waves that are an attractive choice of delivery system, as they oï¬er a non-invasive, targeted and safe approach. Furthermore, the delivery of messenger ribonucleic acid (mRNA) possesses several advantages over commonly delivered plasmid deoxyribonucleic acids (pDNA), because it does not require opening of the nuclear envelope, thereby reducing the level of cell injury necessary for transfection. This work presents the ï¬rst investigation on the eï¬cacy of LSW mediated mRNA delivery, based on optimised SW parameters that balance the desired enhanced permeability of cell membranes against undesired cytotoxicity, and maintain the structural and biological stability of the RNA. A transfectability measure that deï¬nes the ability of SWs to permeabilise a cell whilst keeping it alive was established for dissimilar cell types, as a function of the acoustic pressure and number of SWs. Statistically signiï¬cant RNA uptake was recorded in a tissue mimicking system, and using RNA analogues at various concentrations, the SW induced bio-distribution was characterised. In addition to LSW induced gene augmentation using mRNA, it was shown that LSWs could be used to eï¬ect gene inhibition through the delivery of siRNA. Kinetic experiments were carried out to measure mRNA uptake during shock wave exposure and indicated that rate of delivery was highest at the start of the SW dose and decreased during treatment. The results also suggested that the enhancement of cell permeability was signiï¬cantly transient, and that mRNA was highly susceptible to degradation in its naked state. Furthermore, mRNA-based gene expression was shown to be predictive but quantal. The in vitro tissue model was improved from a gel-based system, to one that incorporated multi-cellular spheroids which capture aspects of 3-D tumours. Static overpressure was applied during SW exposure in order to suppress cavitation eï¬ects and isolate eï¬ects that could be attributed to shear due to cell-to-cell coupling. The results showed that mild overpressure improved RNA uptake the most, but that at higher overpressure, the level of increase in RNA uptake relative to controls, was dependent on the type of RNA nucleotide being delivered. This suggested that a complex interaction between LSW cavitation and direct stress dominates delivery. A ï¬nal report was on the signiï¬cant improvement of gene delivery when mRNA was encapsulated within a lipid nanoparticle vector, and SW exposure was assisted by cavitation agents. Also, by exposure to another acoustic stimulus â focused ultrasound (US), direct comparisons were made between SWs and US on the eï¬ciency of delivery and tissue penetration. In conclusion, this thesis has shown that by choosing parameters appropriately, shock waves can be a promising strategy for the delivery of genes to cells.</p

Acoustically responsive polydopamine nanodroplets: A novel theranostic agent

Ultrasound-induced cavitation has been used as a tool of enhancing extravasation and tissue penetration of anticancer agents in tumours. Initiating cavitation in tissue however, requires high acoustic intensities that are neither safe nor easy to achieve with current clinical systems. The use of cavitation nuclei can however lower the acoustic intensities required to initiate cavitation and the resulting bio-effects in situ. Microbubbles, solid gas-trapping nanoparticles, and phase shift nanodroplets are some examples in a growing list of proposed cavitation nuclei. Besides the ability to lower the cavitation threshold, stability, long circulation times, biocompatibility and biodegradability, are some of the desirable characteristics that a clinically applicable cavitation agent should possess. In this study, we present a novel formulation of ultrasound-triggered phase transition sub-micrometer sized nanodroplets (~400 nm) stabilised with a biocompatible polymer, polydopamine (PDA). PDA offers some important benefits: (1) facile fabrication, as dopamine monomers are directly polymerised on the nanodroplets, (2) high polymer biocompatibility, and (3) ease of functionalisation with other molecules such as drugs or targeting species. We demonstrate that the acoustic intensities required to initiate inertial cavitation can all be achieved with existing clinical ultrasound systems. Cell viability and haemolysis studies show that nanodroplets are biocompatible. Our results demonstrate the great potential of PDA nanodroplets as an acoustically active nanodevice, which is highly valuable for biomedical applications including drug delivery and treatment monitoring