Research has been done on ultrasound-mediated gene transfection for the purpose of developing effective gene therapies. Another study found that ultrasonic-responsive bubble compositions and ultrasound irradiation break the barrier to blood flow to the brain (BBB) and DNA transfer plasmids (pDNA) to the brain by causing cavitation energy to build up inside the membranes. According to a recent study, the degree of BBB permeability may be affected by the amount of echo gas in microbubbles. As a result, doubling the volume of echo gas in bubble composition in the brain would increase their efficiency in transfections. pDNA polyplexes, or cationic macromolecules, have been developed for in vivo gene transfection. They are prone to cause hematotoxicity and cytotoxicity in the circulatory system because of their strong association with blood components and cell membranes. Encapsulation and encapsulation of cationic polyplexes with anionic macromolecules through electrostatic forces have been used to produce biocompatible complexes. On the other hand, these complexes lack the capacity to target cells or tissues specifically. For this, lipo-polyplexes with pDNA were created, cationic polymers, and anionic liposomes that are negatively charged and sensitive to ultrasound (ALS). 

Anions like glucose may be used to avoid the formation of aggregation in binary pDNA/nanobubble systems.  Because of their complex architectures, tertiary bubble lipo-polyplexes synthesized in glucose (5%) have only a modest capability for echo gas (C3F8), indicating that electrostatic forces may be challenging to generate persistent bubble formulations. Following this strategy, bubble lipo-polyplexes with adequate capacity for the encapsulation of C3F8, even though the precise process was unknown, can be produced. Tertiary complexes were made in clean water before being encapsulated in C3F8 in order to maintain a constant concentration of PBS solution. 

In nonionic solutions, the inclusion of electrolytes like NaCl facilitated the formation of stable cationic liposome/pDNA complexes (lipoplexes) with lower sizes, enabling faster membrane fusing between liposomes. It is assumed that the presence of electrolytes like NaCl would alter the production of bubble lipo-polyplexes through the enhanced fusing of membranes. Because of this, the encapsulation effectiveness of C3F8 in bubble lipo-polyplexes may be improved. Because it is hypothesized that C3F8 encapsulation might be achieved using this technology, it was named SCR-EGE (SCR-EGE for short). Using bubble lipo-polyplexes generated by the SCR-EGE technique, we investigated the encapsulation efficiency of C3F8 (SCR-EGE bubble lipo-polyplexes). Because the SCR-EGE-bubble lipo-polyplexes were predicted to enhance C3F8-encapsulation, we administered these bubble lipo-polyplexes to the brain to enable gene transfection. 

Choosing a therapeutic gene based on the location of transgene expression is critical for effective gene therapy in brain disorders. As a result, developing a treatment plan requires knowledge of transgene dispersion. However, there is no such data in the brain employing bubble compositions and ultrasonic irradiation. A tissue-clearing approach and confocal microscopy have been used to construct an observing system for transgenic tissue expression.  Deep imaging is possible after the tissue has been removed. When it comes to determining the location of transgenic expression, this procedure is superior to traditional tissue sectioning. Different tissue-clearing reagents were used in this technique for a variety of applications. For instance, using lipophilic dyes, ClearT2 and ScaleSQ23 may be used to mark biological features such as blood arteries and the peritoneum, while CUBIC21 can be used for deep inspection. 

As a direct result of this, in-depth multicolor imaging of tagged structures, as well as transgenic expression in peritoneal and kidney tissues, was made possible. This multicolor deep imaging technique, which takes into account all of these aspects, was used to clarify the 3D distribution of transgene expression and brain blood arteries. SCR-EGE bubble lipo-polyplexes were produced and their physical and chemical properties were evaluated to see if they helped with C3F8 encapsulation. After that, fluorescence resonance energy transfer was used to gauge the degree of membrane fusion (FRET). This was then proceeded by an examination of transgenic expression during brain ultrasound irradiation in mice treated with SCR-EGE bubble lipo-polyplexes. In addition, deep multicolor imaging with ScaleSQ elucidated the 3D pattern of transgenic expression in the cerebral cortex. As a way to achieve long-term expression in organs like the lungs and liver, it was also tested whether CpG-depleted vectors might be used to maintain transgene expression in the brain. 

To begin, it looked at how well C3F8 could be encapsulated in SCR-EGE bubble lipo-polyplexes. The transfection efficacy of these bubble lipo-polyplexes was then assessed in mice brains. Multicolour deep imaging was also used to track the spread of transgenes in three dimensions. Compared to normal bubble lipo-polyplexes, SCR-EGE bubble lipo-polyplexes had a higher C3F8 concentration. The anionic potential of SCR-EGE bubble lipo-polyplexes was demonstrated, as was the absence of clumping with erythrocytes. Using SCR-EGE bubble lipo-polyplexes and ultrasonic irradiation, high transgenic expression was found. Since SCR-EGE bubble lipo-polyplexes were found mostly on blood vessels, transgene expression was also seen in the exterior of blood vessels. 

When using bubble lipo-polyplexes with a CpG-depleted vector to transfect the brain, it was determined how long the transgene expression lasted. For at least 28 days, pCpG-depleted-Luc was shown to be the primary driver of transgene expression. After only seven days, pCMV-Luc transgene expression dramatically dropped. There were no significant differences between mice receiving SCR-EGE bubble lipopolyplexes and those receiving conventional bubble lipo-polyplexes when it came to transgene expression. 

Using SCR-EGE bubble lipo-polyplexes with a CpG-depleted vector, these results show that strong transgenic expression may be maintained. Researchers have discovered that SCR-EGE bubble lipo-polyplexes, which contain pDNA depleted of CpG and, as a result, release therapeutic proteins for longer periods of time, are an effective method for transfecting blood vessels and delivering therapeutic proteins. SCR-EGE bubble lipo-polyplexes containing C3F8 have been proven to promote bubble lipo-polyplexes containing C3F8 and to be useful carriers for safe and successful brain transfection in combination with ultrasound. Scientists were able to map out the three-dimensional distribution of transgenic translation in brain tissue, with considerable transgene expression seen on blood vessels. For example, bubble lipo-polyplexes can be treated with ultrasonically irradiated lipo-polyplexes.