Overview of Photoporation-based Gene Delivery

Gene delivery refers to the technology of effectively transferring exogenous genes or drugs into target cells or tissues, thereby changing their phenotype or function. Gene delivery has broad application prospects in biomedical research and clinical treatment, especially for some inherited or acquired diseases that are difficult to treat with traditional drugs, such as cancer, neurodegenerative diseases, infectious diseases, etc. However, gene delivery also faces many challenges, such as low delivery efficiency, high cytotoxicity, strong immune response, and poor tissue targeting. To overcome these challenges, a variety of gene delivery methods have been developed, including physical, chemical, and biological methods. Among them, the physical method refers to using the principles of physical mechanics or electromagnetics to directly or indirectly form pores on the cell membrane, so that foreign genes or drugs can enter the cells. Physical methods have the advantages of wide application range, simple operation, and non-immunogenicity, but they also have disadvantages such as low delivery efficiency, large cell damage, and difficulty in in vivo delivery. In recent years, a new type of physical method—photoporation has attracted widespread attention. Photoporation refers to the use of laser or other light sources to irradiate cells, thereby creating pores in the cell membrane, allowing foreign genes or drugs to enter the cells. Photoporation has the advantages of high delivery efficiency, low cytotoxicity, flexible operation, and in vivo delivery. Photoporation can be divided into different categories according to different types of light sources and mechanisms of action.

Fig.1 Schematic Overview of The Most Commonly Used Photoporation Protocol

Types and Mechanisms of Photoporation

Photoporation refers to the use of laser or other light sources to irradiate cells, thereby creating pores in the cell membrane, allowing foreign genes or drugs to enter the cells. Photoporation can be divided into different categories according to different types of light sources and mechanisms of action, such as photoacoustic photoporation, photothermal photoporation, and photoelectric photoporation. Photoacoustic photoporation refers to the use of pulsed lasers to irradiate cells containing photothermal conversion materials (such as gold nanoparticles and carbon nanomaterials), thereby generating vapor nanobubbles around the cells. The rapid expansion and contraction of the bubbles exert mechanical forces on the cell membrane, resulting in Cell membrane rupture or pore formation. Photoacoustic photoporation has the advantages of high delivery efficiency, low cytotoxicity, flexible operation, and in vivo delivery. Photothermal photoporation refers to the use of continuous laser light to irradiate cells containing photothermal conversion materials, thereby generating a local temperature rise around the cells, which leads to a phase change or fluidity change of the cell membrane, thereby allowing foreign genes or drugs to enter the cells. Photothermal photoporation has the advantages of high delivery efficiency, simple operation, and in vivo delivery. Photoelectric photoporation refers to the use of pulsed lasers to irradiate cells containing photoelectric conversion materials (such as strontium barium titanate nanoparticles), thereby generating a local electric field around the cells, and the electric field applies a voltage to the cell membrane, resulting in cell membrane electroporation or pore formation. Photoelectric photoporation has the advantages of high delivery efficiency, simple operation, and in vivo delivery. The main influencing factors of these three kinds of photoporation include laser parameters (such as wavelength, power, pulse width, repetition frequency), the characteristics of photoelectric conversion materials (such as type, size, shape, concentration), and the characteristics of cells (such as type, size, shape, quantity).

Table 1. Comparison of different types of photoporation methods for intracellular delivery

Application of Photoporation in Disease Treatment

Photoporation, as an efficient, safe, and flexible gene delivery method, has broad application prospects in disease treatment. Photoporation can deliver different types of genes or drugs, such as DNA, RNA, proteins, nanoparticles, etc., to achieve functions such as gene editing, gene silencing, gene expression, and drug release. Photoporation can also be delivered to different types of cells or tissues, such as blood, liver, nervous system, retina, thereby achieving tissue targeting and selectivity. Photoporation can also treat different types of diseases, such as genetic diseases, tumors, infectious diseases, so as to achieve cure or remission.

DNA is a common gene delivery vehicle that can be used for purposes such as gene editing, gene expression, or gene therapy. Photoporation can efficiently deliver DNA into cells while maintaining its integrity and functionality. For example, using photoacoustic photoporation, plasmid DNA can be delivered into human embryonic stem cells (hESCs) and achieve high transfection efficiency and high survival rate, while maintaining the pluripotency and differentiation potential of hESCs. Using photoelectric photoporation, plasmid DNA can be delivered into human fibroblasts (hDFs) and achieve high transfection rate and high survival rate, while inducing the reprogramming of hDFs to induce pluripotent stem cells (iPSCs). RNA is used as a gene delivery vehicle for purposes such as gene silencing, gene expression, or gene therapy. Likewise, photoporation can efficiently deliver RNA into cells while maintaining its stability and functionality. For example, by using photoacoustic photoporation, mRNA or siRNA can be delivered into human T cells, and achieve high transfection rate and high survival rate, while realizing T cell receptor (TCR ) or programmed death protein 1 (PD1) expression or silencing. Using photoelectric photoporation, CRISPR-Cas9 RNP can be delivered into human retinal pigment epithelial cells (hRPEs), and achieve high transfection rate and high survival rate, while achieving knockout of the retinol binding protein 4 (RBP4) gene. In the same way, proteins are used for purposes such as drug delivery or bioimaging. Utilizing photoacoustic photoporation, fluorescent proteins or antibodies can be delivered into human liver cancer cells (HepG2), and achieve high transfection rate and high survival rate, while achieving fluorescent labeling or antigen identify. Using photoelectric photoporation, fluorescent proteins or enzymes can be delivered to human lung cancer cells (A549), and achieve high transfection rate and high survival rate, and at the same time realize fluorescent labeling or catalytic reaction. Finally, nanoparticles are a novel gene delivery vehicle that can be used for purposes such as drug delivery or bioimaging. Using photoacoustic photoporation, quantum dots or ferrite nanoparticles can be delivered into human liver cancer cells (HepG2), and achieve high transfection rate and high survival rate, while achieving Fluorescence labeling or MRI. Moreover, the use of optoelectronic photoporation can deliver gold nanorods or carbon quantum dots into human lung cancer cells (A549), and achieve high transfection rate and high survival rate, while achieving Surface enhanced Raman scattering or fluorescence imaging.

Challenges and Prospects

Although photoporation has shown great potential in disease treatment, there are still some problems and challenges that require further research and improvement.

Firstly, the light source is one of the key factors of photoporation, which affects the efficiency, safety and applicability of photoporation. At present, commonly used light sources include ultraviolet light, visible light, near-infrared light, and near-infrared light, each with its own advantages and disadvantages. Ultraviolet light has high energy and can directly affect cell membranes, but it can also cause damage to cellular DNA and cell death. Visible light has lower energy and can reduce cell damage, but is also limited by tissue scattering and absorption. Near-infrared light has good tissue penetration and can realize photoporation of deep tissues, but it will also cause high tissue temperature rise and thermal damage. Near-infrared light in the second region has better tissue penetration and can achieve photoporation of deeper tissues, but it also requires higher power lasers and more stable photosensitive materials. Therefore, how to select and optimize a suitable light source to achieve efficient, safe, flexible, and economical photoporation is an urgent problem to be solved.

Secondly, the photosensitive material is also one of the key factors of photoporation, which affects the efficiency, safety and functionality of photoporation. At present, commonly used photosensitive materials include metal nanoparticles, carbon nanomaterials, semiconductor quantum dots, organic dyes, each with its own advantages and disadvantages. Metal nanoparticles have high photothermal conversion efficiency and can realize photoacoustic or photothermal photoporation, but they also cause problems of biocompatibility and biosafety. Carbon nanomaterials have good biocompatibility and biodegradability, and can realize photoacoustic or photothermal photoporation, but they are also affected by purity and uniformity. Semiconductor quantum dots have excellent fluorescence performance and adjustable emission wavelength, which can realize fluorescence imaging or photoelectric photoporation, but there are also problems of toxicity and stability. Organic dyes have the advantages of low cost and easy synthesis, and can realize fluorescence imaging or photoelectric photoporation, but they also encounter problems such as fluorescence quenching and photobleaching. Therefore, it is necessary to figure out how to design and improve suitable photosensitive materials to achieve efficient, safe, functional, and intelligent photoporation.

Thirdly, cell type and state are also critical for photoporation, affecting the efficiency, safety and applicability of photoporation. At present, many studies have shown that cells of different types or states have different responses to photoporation. For example, attached cells are more susceptible to photoporation than suspension cells; stem cells are more sensitive to photoporation than differentiated cells; tumor cells are more susceptible to photoporation than normal cells; T cells in an activated state are more susceptible to photoporation than T cells in a resting state, and so on. These differences may be related to factors such as cell membrane structure, composition, fluidity, and charge. Therefore, the differential response of cells of different types or states to photoporation should be considered and exploited.

Finally, in vivo application and clinical transformation are one of the ultimate goals of photoporation, which is of great significance in disease treatment. At present, some studies have shown that photoporation has been successfully applied in animal models to treat tumors, hereditary retinal degenerative diseases. However, there are still many challenges in the process of in vivo application and clinical translation. For example, there are complex and variable physiological parameters, biological barriers, biological distribution, biological responses and other factors in the internal environment. These factors will affect the consideration of laser parameters, photosensitive material properties, gene or drug properties, when photoporation is applied in vivo, and may lead to unpredictable or uncontrollable effects on the effect of photoporation in vivo application. Therefore, in the process of in vivo application and clinical transformation, a large number of rigorous and systematic basic and preclinical experiments are required, and relevant regulations and standards need to be followed to ensure its effectiveness and safety.

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