For gene delivery in difficult to transfect cells, scholars from the National University of Singapore have developed a chimeric gene delivery system using Microcellular Vesicle Technology (mCVT). This study utilized a liposome extruder from the American company Genizer and published the results in the journal Nanoscale. The full name of the original text is: Micro cell vesicle technology (mCVT): a novel hybrid system of gene delivery for hard-to-transfect (HTT) cells. DOI

In this study, a research team from the National University of Singapore introduced a novel chimeric gene delivery platform system based on micro cell vesicle technology (mCVT). This gene delivery platform system is composed of cell membrane and cationic lipid fusion, and is used to improve the transfection efficiency of hard to transfect HTT cells. The cell membrane components of microbubbles endow the entire complex with phagocytic specificity, reducing the cytotoxicity of the complex during transfection, while cationic lipids are responsible for binding to genetic material and providing structural integrity for mCVTs.

background

Gene delivery is the process of introducing foreign DNA into eukaryotic cells. Such as delivering therapeutic or functional genes to alter cellular processes in basic research (such as drug discovery and mechanism research of functional proteins) and clinical applications (such as gene therapy). Effective gene transfection not only requires the successful introduction of foreign nucleic acids into cells, but also their subsequent transport and expression within the cells. This is a challenging task because DNA molecules themselves are negatively charged, making it difficult for them to be internalized by cells in their conventional form. Therefore, people have shown great interest in developing effective gene delivery systems. In the past few decades, a large number of vector systems have been explored, ranging from virus based delivery systems to non viral vectors. Although the transfection efficiency of non viral gene delivery vectors is relatively low, they have the potential to become safe and clinically acceptable, with cationic lipid systems and polymer based platforms being the main examples. Table 1 summarizes the main advantages and disadvantages of commonly used transfection reagents or methods.

An ideal non viral gene delivery system should be able to effectively deliver the required DNA molecules to specific cell types with minimal cytotoxicity to ensure maximum expression of the target gene. However, to our knowledge, currently available transfection reagents clearly do not meet these standards, especially when they are used to transfect difficult to transfect (HTT) cells.

HTT cells typically include primary cells, embryonic stem cells, and some immortalized cell lines, such as 3T3-L1 (mouse preadipocytes), U937 (human monocytes), Jurkat (human T lymphocytes), HUVEC (human umbilical vein endothelial cells), etc. According to statistics, the efficiency of plasmid transfection of HTT cells using commercially available reagents often does not exceed 25%. One of the reasons for the low absorption rate of foreign substances and plasmids by HTT cells is their low metabolic activity. In addition, these cells are often highly sensitive to cell disturbances caused by the transfection reagents used, which may lead to cytotoxicity and ultimately result in cell death.

Recently, cell derived delivery systems have received considerable attention due to their potential to selectively deliver active cargo molecules to target sites to a certain extent. In fact, many studies have explored the use of monocyte derived drug delivery systems (DDS) for targeted delivery of chemotherapy drugs and plasmid DNA (pDNA) to tumors, or red blood cell-derived DDS for prolonging blood circulation and targeted delivery of pDNA to blood cells. It is speculated that retaining the cell membrane components of the mother cells will endow these cell-based drug delivery systems with the ability to recognize specific target cells, possibly by evading the recognition ability of the body's immune system.

Despite its significant therapeutic implications, the full potential of cell-based DDS in gene delivery has yet to be fully exploited. The key challenges that need to be addressed include the tedious and low throughput separation/production process of cell-derived DDS (such as in the form of exosomes), as well as suboptimal gene loading/recombination technology. Therefore, although these systems seem to have advantages in terms of intrinsic targeting, they have significant limitations in terms of yield and pDNA loading efficiency. Therefore, in order to overcome these limitations, we propose to develop a novel chimeric gene delivery system consisting of cell membrane components and cationic lipid components. The former is used to enhance the internalization of target cells, while the latter is used to enhance the loading of pDNA.

In this study, we described the development of Microcellular Vesicle Technology (mCVTs), a novel hybrid delivery platform obtained by fusing cationic lipids with cell membranes. To produce mCVTs, cells were first treated with a low osmotic solution to obtain ghost cells (CG without cytoplasm in organelle cells), as described by Goh et al. Low osmolarity solution forms brief openings on the plasma membrane, allowing for the clearance of intracellular contents while maintaining the integrity of the membrane. CG from different cells suspended in an aqueous medium was used to hydrate and dry DOTAP lipid films, which were then extruded using a liposome extruder to provide mCVTs with average hydrodynamic dimensions and Zeta potential similar to the extruded DOTAP liposomes. In this study, we demonstrated that mCVTs can complex with plasmids and be internalized by target cells with minimal cytotoxicity. Interestingly, when comparing mCVTs produced from different cell types in vitro, they showed significantly higher transfection efficiency for the cell types from which mCVTs originated.

Method and Results

Prepare ghost shadow cells (CG without cytoplasm and organelle cell membrane) to ensure the reproducibility and safety of mCVTs.

Method and Results Brief

Production and feature analysis of mCVTs.

Similar to liposome production, lipid film method is used to produce mCVTs. DOTAP film hydrates with CG to form large multi molecular liposomes encapsulating CG. This suspension was extruded, and the plasma membrane from CG contained DOTAP lipids (as shown in Figure 3A). According to the method previously established by our group, it has been demonstrated that after compression, the resulting mCVTs are composed of the fusion of lipids and CG membranes. Generally speaking, the size distribution of mCVT is similar to that of liposomes. To demonstrate the robustness and universality of this method in various cell lines, mCVTs were fabricated using CG from different cell lines, such as some HTT cells (i.e. 3T3-L1) and non cancer cells (i.e. HEK293 and RAW264.7). As shown in Figure 3B, the hydrodynamic dimensions and PDI of various mCVTs are typically in the range of 400 nanometers (PDI~0.4), similar to DOTAP liposomes produced using the same extrusion method (PDI~0.2). In Figure 3C, similar zeta potential values (approximately+30 mV to+40 mV) were observed for different formulations, indicating that this method is generally applicable to CG produced from different cell lines. Interestingly, when mCVT3T3-L1 is complexed with pDNA, its size distribution is similar to that of uncomplexed mCVTs, while the overall zeta potential becomes negative, which may indicate successful complexation (as negatively charged pDNA neutralizes positively charged mCVTs).

A. Schematic diagram of mCVTs, involving the fusion of cell ghosts and liposomes made from hydrated membranes.

B. The hydrodynamic dimensions of different mCVTs and mCVT3T3-L1 with plasmids were compared to DOTAP liposomes. All mCVTs have a size less than 500 nanometers.

C. Compared with DOTAP liposomes, the Zeta potential of different mCVTs and plasmid carrying mCVT3T3-L1. The Zeta potential of all mCVTs is above+30 mV, except for mCVT3T3-L1 with plasmid DNA (5 µ g), which exhibits a negative Zeta potential due to interaction with plasmid DNA. The data represents the mean ± SD (n=3).