A mini-review on gene delivery technique using nanoparticles-mediated photoporation induced by nanosecond pulsed laser

Abstract

Nanosecond pulsed laser induced photoporation has gained increasing attention from scholars as an effective method for delivering the membrane-impermeable extracellular materials into living cells. Compared with femtosecond laser, nanosecond laser has the advantage of high throughput and low costs. It also has a higher delivery efficiency than continuous wave laser. Here, we provide an extensive overview of current status of nanosecond pulsed laser induced photoporation, covering the photoporation mechanism as well as various factors that impact the delivery efficiency of photoporation. Additionally, we discuss various techniques for achieving photoporation, such as direct photoporation, nanoparticles-mediated photoporation and plasmonic substrates mediated photoporation. Among these techniques, nanoparticles-mediated photoporation is the most promising approach for potential clinical application. Studies have already been reported to safely destruct the vitreous opacities in vivo by nanosecond laser induced vapor nanobubble. Finally, we discuss the potential of nanosecond laser induced phototoporation for future clinical applications, particularly in the areas of skin and ophthalmic pathologies. We hope this review can inspire scientists to further improve nanosecond laser induced photoporation and facilitate its eventual clinical application.

Keywords:

• Drug delivery
• photoporation
• nanosecond pulsed laser
• nanobubble

1. Introduction

Intracellular delivery of exogenous substances into cells has become a hotspot issue in the field of biological research (Ramon et al., 2021). Foreign substances, such as RNA, genome editing complex, plasmid DNA, antibodies, and nanoparticles, are often delivered into living cells to confer the outcome of cell-based therapies, gene edited cells, induced pluripotent stem cells, control gene expression and probing the intracellular environment (Stewart et al., 2018). Based on these applications, it is promising for the treatment of genetic diseases and illnesses that cannot be cured with conventional drugs (Du X et al., 2018). Small molecules are often taken up by cells through passive diffusion or endocytosis (Schneckenburger, 2019). Nevertheless, most of the foreign substances are membrane-impermeable. Therefore, it is essential for intracellular delivery to overcome the barriers of cell membrane.

In order to achieve effective drug delivery, much effort has been put into the study of intracellular delivery technique, such as biological, chemical, mechanical and physical technique. The advantages and disadvantages of these delivery techniques are summarized in Table 1. Viral vector-based biological delivery system has been successfully applied in clinical research (Pasi et al., 2020; Esrick et al., 2021; Kohn et al., 2021; Paunovska et al., 2022), which is high-efficiency and easy to use (Kim & Eberwine, 2010). However, it is constrained by some factors, including viral-induced immunogenicity (Xiong et al., 2016; Ramon et al., 2021), payload size constraints (Wu et al., 2010) and expensive vector production (Shinde et al., 2021; Paunovska et al., 2022) etc. Considering its limitations, non-viral chemical vectors, such as polymers, lipids, and inorganic materials, are designed for intracellular delivery (Stewart et al., 2016; 2018; Shinde et al., 2020). Compared to biological methods, chemical methods are safer and less expensive. However, the new drawbacks have emerged, such as low delivery efficiency, poor endosomal escape, toxicity of chemical vector and low instability, (Varkouhi et al., 2011; Yadav et al., 2017; Hosseinpour & Walsh, 2021). Therefore, the mechanical approaches like microinjection, nanoneedles and gene gun are applied to directly penetrate the cell membrane (Du X et al., 2018; Stewart et al., 2018; Duckert et al., 2021) and create a passage for foreign substances. Mechanical methods are the simplest and most straightforward technique, and have a good delivery efficiency (Gao et al., 2007; Kim & Eberwine, 2010). The first intracellular delivery approach of microinjection is invented in 1911 by Marshal Barber (Barber, 1911). It can be used for delivering almost any cargo, but the low throughput constrains its application. Nanoneedles has a similar principle with microinjection, but the complex equipment limits its application in vivo (Chiappini et al., 2015). Gene gun shots the cargo into cells using pressurized gas (Klein et al., 1992), however it may damage the cell (Zhang et al., 2014).

Table 1. Advantages and disadvantages of current delivery techniques.

Class		Disadvantage	Advantage
Biological		Viral-induced immunogenicity; Payload Size constraints; Expensive	High-efficiency; Easy to use
Chemical		Low delivery efficiency; Poor endosomal escape; Low instability; Toxicity	Safer and less expensive than biological vector
Mechanical		Need special instrument; Low throughput; May damage cell	Simplicity and straightforward; Good efficiency
Physical	Electroporation	Low cell viability; Intricate electrodes	High delivery efficiency
	Sonoporation	Low delivery efficiency; Poor controlled precise	Less invasive
	Magnetoporation	Low delivery efficiency; Agglomeration of magnetic nanoparticle	Noninvasive
	Photoporation	High cost of laser and optical system	Noncontact; Contamination-free; Less dependent on cell type

Different with above methods, physical methods disrupt cell membrane through physical energy, which have been successfully used for intracellular delivery. Electroporation (Santra et al., 2013; Kar et al., 2018), sonoporation (Wang et al., 2018; Karki et al., 2019), magnetoporation (Liu et al., 2012) and photoporation (Ramon et al., 2021) are the main physical approaches. Compared with viral and chemical methods, these approaches enables the delivery of cargo into any type of cell while being not-toxic (Du X et al., 2018; Shinde et al., 2020). However, each method has its disadvantages and advantages. It is well known that electroporation can acquire high delivery efficiency, however, low cell viability limits its application (Gao et al., 2007; Mellott et al., 2013). In addition, intricate electrodes must be designed for different application scenarios (Heller & Heller, 2006). Sonoporation is less invasive and has been used in clinical setting (Mellott et al., 2013; Lakshmanan et al., 2014). However, its low delivery efficiency and poor controlled precise within the tissue limit its application (Mehier-Humbert & Guy, 2005; Mellott et al., 2013). Magnetoporation is a noninvasive technique that delivers the foreign cargo under magnetic field. Generally, exogenous cargo needs to bind with magnetic nanoparticles. Thus, the agglomeration of magnetic nanoparticles after removal of the magnetic fields limits its application (Du X et al., 2018). In addition, poor delivery efficiency is still a problem to be resolved (Mehier-Humbert & Guy, 2005). Photoporation is another physical technique, which also sometimes called optoporation (Yao et al., 2020), optical injection (Duckert et al., 2021), optoinjection (Krasieva et al., 1998), laserfection (Rhodes et al., 2007) or optical transfection (Stevenson et al., 2010). Photoporation is the technique using laser to penetrate and create pore on cell membrane, allowing exogenous cargo to diffuse into cells based on their concentration gradient. A breakthrough work was reported by Tirlapur and König until 2002 (Tirlapur & König, 2002). They transfected green fluorescent protein (GFP) using a near-infrared femtosecond laser. By now, it has been widely used for intracellular delivery due to the merits of noncontact, contamination-free and less dependent on cell type (Kim & Eberwine, 2010; Shinde et al., 2021). Moreover, it has been widely used to transfect kinds of cargo, such as DNA, siRNA, mRNA, protein, and nanoparticle (Stewart et al., 2018). However, the cost of laser and optical system is high.

Laser source is an important factor affecting photoporation. Generally, three modes of laser, continuous-wave (CW) laser, femtosecond pulsed (FS) laser, and NS laser, are used for photoporation (Gu et al., 2014; Minai et al., 2016; Van Hoecke et al., 2019; Hosseinpour & Walsh, 2021). NS laser is widely used for intracellular delivery in recent years. Therefore, this review offers an overview of NS laser induced photoporation technique for intracellular delivery. The perforation mechanism of NS laser is presented in the initial section. This is followed by a discussion of the existing approaches of photoporation induced by NS laser. Factors that affect delivery efficiency are then described. Finally, the latest advances in NS laser-induced photoporation are highlighted, along with a discussion of the prospects and challenges for using nanosecond pulsed lasers induced photoporation.

2. The mechanism of photoporation

The mechanism of photoporation is complex and it varies depending on laser type. CW laser perforates the cell membrane mainly based on the localized heating (Schneckenburger et al., 2002; Stevenson et al., 2010). It will increase the temperature of cell membrane, which will induce the change of membrane dynamic. A phase transition of gel-to-liquid-crystalline is formed in lipid bilayer membrane (Chapman et al., 1995). Thus, the permeability of cell membrane is changed, and the exogenous materials are more easily to enter into cell (Schneckenburger, 2019). For CW laser, it delivers materials by changing the permeability of cell membrane. Therefore, its delivery efficiency is not satisfactory. The CW laser can only obtain a low delivery efficiency less than 30% (Stevenson et al., 2010). In addition, the process of photoporation by CW laser is time-consuming, which often takes up a few minutes to change the permeability of cell membrane (Schneckenburger et al., 2002; Xiong et al., 2016).

For FS laser, the mechanism is based on the nonlinear absorption phenomenon, which generates the low-density plasma to penetrate the cell membrane (Vogel et al., 2005; Stevenson et al., 2010). On the other hand, it can generate vapor nanobubble (VNB) induced by low-density palsma to create pore on cell membrane (Stevenson et al., 2010; Shinde et al., 2020). Expansion and collapse of VNBs can create mechanical stress which can destroy the cell membrane and open a single pore. Baumgart el al. transfected human cancer melanoma cells based on the FS laser induced cavitation bubble and obtained a high delivery efficiency of 70% (Baumgart et al., 2012). In addition, FS laser can breakdown the organic molecular bonds and lead to membrane disruption, which is called photochemical effect (Srinivasan & Leigh, 1982; Srinivasan, 1986). Meunier’s team has studied the FS laser induced photoporation for a long time (Baumgart et al., 2012; St-Louis et al., 2013; Bergeron et al., 2015; Lachaine et al., 2016). In their latest research, they reported an in vivo method to perforate retinal ganglion cells using FS laser, which had potential to cure retinal degenerative diseases (Wilson et al., 2018).

NS laser is another commonly used pulsed laser in recent years. The mechanism of NS laser mainly based on heating, VNBs and thermoelastic mechanical stress (Stevenson et al., 2010; Stewart et al., 2018). As same as CW laser, it also changes the permeability of cell membrane by heating effect when laser energy density below the threshold of VNBs formation. However, the delivery efficiency is not ideal caused by NS laser induced photothermal effect (Xiong et al., 2014). Once the photothermal nanoparticles are exposed in a high enough laser energy density, it will cause VNBs formation (Figure 1). In this scenario, the mechanism of photoporation mainly attributes to photomechanical effects. Liquid jets and shockwave will be generated in the process of VNBs expansion and collapse, which can form a pore in cell membrane (Xiong et al., 2016). On the other hand, acoustic wave will be generated after absorbing heat by irradiated materials, which will cause the thermoelastic stress. It can cause tensile stress and form VNBs, which can lead to cell perforation. For nanoparticles-mediated NS laser induced photoporation, the mechanism of photoporation mainly attributes to the thermophysical responses of the top parts in the Figure 1 (Green arrow). In addition, it may generate plasma when the laser energy is stronger enough, and plasma-induced VNBs will be generate (Black arrow in Figure 1). However, this effect is minimal for NS laser in presence of nanoparticles. Plasma-induced VNBs often generates by a high-intensity FS laser via multiphoton ionization.

Figure 1. Schematic for response of laser induce photothermal nanoparticle. Created by the author Xiaofan Du.

The mechanism of VNBs generation is different between NS and FS laser, which is detailed in the publications by Xiong et al. and Vogel et al. (Vogel et al., 2005; Xiong et al., 2016). Moreover, the VNBs induced by NS laser is larger and more violent than FS laser, and the pore formed in cell membrane is also larger. Therefore, NS laser is more appropriate for delivering large molecule.

In conclusion, the mechanism of photoporation is different among CW, FS and NS laser. The delivery efficiency of diverse laser is also different. The delivery efficiency of CW laser is lower than NS laser and FS laser. FS laser can obtain high delivery efficiency, but its equipment is very expensive and it is more suitable for manipulation of single cell (Heinemann et al., 2013). NS laser, as a compromise, can provide high delivery efficiency at a lower cost than FS laser (Hosseinpour & Walsh, 2021; Ramon et al., 2021). In addition, NS laser can acquire high-throughput intracellular delivery with the help of nanoparticles. Therefore, NS laser is an ideal option for photoporation.

3. The way of nanosecond pulsed laser induced photoporation

3.1. Direct nanosecond laser induced photoporation

Direct photoporation is a method in which a laser beam is applied directly to the cell membrane, generating transient pores. In 1984, NS laser was employed directly for transfecting DNA. Tsukakoshi et al. presented a new technique in their report, in which foreign DNA was delivered into normal rat kidney cell using a NS laser with a wavelength of 355 nm and a duration time of 5 ns (Tsukakoshi et al., 1984). In 2006, Clark et al. firstly demonstrated that the photoporation was an automated and high throughput method to deliver foreign materials directly (Clark et al., 2006). Various substrates including ions, small molecules, dextrans, plasmids, siRNA, proteins, and nanocrystals have been delivered into different cells. However, the delivery efficiency of this method was not satisfactory, as the laser energy threshold causes plasma formation is extremely sharp (Vogel et al., 2005). Hence, it often leads to a high plasma or no plasma at all, which may result in high cell death ratio or low delivery efficiency.

3.2. Nanoparticles-mediated nanosecond laser photoporation

In order to improve the delivery efficiency and cell viability, various nanoparticles, such as gold nanoparticles (St-Louis et al., 2013; Bergeron et al., 2015; Teirlinck et al., 2018), carbon nanoparticles (Sengupta et al., 2014), and graphene quantum dots (Liu et al., 2018), are used to mediate NS laser induced photoporation. Gold nanoparticles are the most commonly used mediator, such as gold nanosphere, gold nanorods, and gold nanosphere shells (Lachaine et al., 2016; Raes et al., 2019; Yao et al., 2020). Laser induced heat and VNBs are the main factors induced photoporation. Xiong et al. demonstrated that the VNBs can deliver the foreign materials more efficiently than gold nanoparticle laser heating (Xiong et al., 2014). It mainly perforates cell by laser induced heat for direct NS laser induced photoporation, while VNBs play dominant role for nanoparticles-mediated nanosecond laser photoporation. Therefore, the participation of nanoparticles can significantly improve the delivery efficiency. In addition, the use of nanoparticle can reduce the required dose of laser energy density, which helps to ensure cell viability. Qin et al. provided a detailed explanation of the mechanism by which laser induces heating and VNBs (Qin & Bischof, 2012), which serves as a reference for the study of photoporation. Due to nanoparticle can significantly improve delivery efficiency of photoporation, nanoparticles-mediated nanosecond laser photoporation has been widely studied by several research teams.

Our group have focused on gold nanoparticles-mediated photoporation since 2005 (Yao et al., 2005; 2009). We demonstrated that the permeability of cell membrane can be elevated by NS laser irradiating gold nanoparticles, which could be used for gene delivery. Subsequently, we studied the impact of laser parameters, such as laser energy density, pulse duration, exposure time, and irradiation mode, on photoporation (Yao et al., 2009). The results showed that antibodies can be delivered into living cells after adjusting the laser parameters. Furthermore, we investigated the effect of other parameters, such as cell-nanoparticle incubation time, and nanoparticle concentration, on photoporation (Yao et al., 2017). It was further used to transfect adherent and trypsinized cells, in which trypsinized cells were more likely to be transfected. In our recent research, proteins were delivered into living cells by antibody modified gold nanorods-mediated photoporation (Yao et al., 2020).

Heinemann et al. had also studied the technique of gold nanoparticle-mediated laser transfection, but they used a picosecond pulsed laser. They delivered fluorescent labeled siRNA into canine pleomorphic adenoma ZMTH3 cells to knock down gene. Furthermore, they have studied the influence factors of photoporation and the mechanisms of molecule uptake (Kalies et al., 2014). They demonstrated that multiphoton ionization of water and thermal effects were the underlying perforation mechanism. They showed that the cell medium was an important factor for delivery efficiency, which was consistent with our previous research (Yao et al., 2017). They also delivered the Caspase 3 into cells to induce apoptotic cell death with high throughput (Heinemann et al., 2014).

Another famous team that studied nanoparticles-mediated NS laser induced photoporation is Kevin Braeckmans’s team. In 2014, they firstly demonstrated that the delivery of foreign materials under conditions of NS laser induced VNBs was much more efficient than laser heating of gold nanoparticles (Xiong et al., 2014). Then, they employed VNBs induced photoporation to resolve some biomedical problems. They delivered contrast agents into cytoplasm for cell imaging with high throughput and low toxicity, and could achieve loading efficiency up to 50 times and 3 times higher for fluorescent dextrans and quantum dots, respectively (Xiong et al., 2016). They demonstrated for the first time that delivering the labels directly into cytosol by photoporation can avoid asymmetric inheritance of labels. They also disrupted the nuclear envelope by VNBs mediated photoporation to deliver the membrane-impermeable macromolecules into cell nucleus (Houthaeve et al., 2018). Meaningfully, they disrupted biofilms by VNBs mediated photoporation to improve diffusion in biofilms of antibiotics and antimicrobial agents, which can be used to resolve antibiotic resistance and wound care (Teirlinck et al., 2018; 2019). Further, they developed various nanoparticles, including layer-to-layer nanoparticles, nanostars layers, graphene, black phosphorus, and biodegradable polydopamine nanosensitizers, to mediate photoporation (Liu et al., 2018; Pylaev et al., 2019; Liu et al., 2020; Shaabani et al., 2021; Wang et al., 2021; Harizaj et al., 2021c). In addition, Kevin Braeckmans’s team has reported some excellent photoporation techniques to address biomedical issues (Sauvage et al., 2019; Van Hoecke et al., 2019; Xu et al., 2020; Raes et al., 2021; Harizaj et al., 2021a; 2021b). In their recent work, NS laser induced VNBs was employed to disrupt vitreous opacities. The in vivo experiments were performed in rabbit eye, which confirmed the safety and effectiveness of NS laser. It shows the potential of NS laser induced VNBs for intracellular delivery, which opens up the possibility of VNBs mediated photoporation technique toward clinical application (Sauvage et al., 2022).

3.3. Plasmonic substrates mediated nanosecond laser induced photoporation

Different from nanoparticle-mediated photoporation, various nanostructured substrates were designed to mediate NS laser induced photoporation. Living cells were incubated onto this substrates, and a NS laser was employed to irradiate it and deliver foreign materials into the cells. This technique aimed to obtain a high-throughput result and resolve the cytotoxicity of nanoparticles.

Mazur’s team designed a pyramid structure plasmonic substrates with a gold thin film on top to acquire intracellular delivery (Figure 2A and B). Delivery efficiency of 95% and cell viability of 98% were obtained for calcein green with a molecule weight of 0.635 kDa. Meanwhile, 50 000 cells were treated in one minute (Saklayen et al., 2017). They further designed another titanium nanocavity substrates shown in Figure 2(C and D), which could acquire a delivery efficiency of 78% and a throughput of 30 000 cells per minute while maintaining a cell viability of 87% (Madrid et al., 2018). Another famous team is Pei-Yu Chiou’s team. They designed a platform aimed to deliver large cargos into cells (Wu et al., 2015). A wide range of cargo types, bacteria, enzymes, antibodies, and nanoparticles, were successfully delivered into different cells, while maintaining a high throughput of 100 000 cells per minute. In another report, they designed a sharp nanoscale metal-coated tips that positioned on microwells to transfect suspension cells (Figure 2E and F) (Man et al., 2019). A delivery efficiency of >84% and >45% can be obtained for calcein green (0.6 kDa) and FITC-dextran (2000 kDa), respectively. Meanwhile, a throughput of >100 000 cells per minute were obtained. In addition, Santra et al. also designed some plasmonic substrates, such as titanium microdish device, TiO₂ micro-flowers substrate and nano-spiked gold nanoparticles substrate, to mediate photoporation (Shinde et al., 2020; Gupta et al., 2021; Mohan et al., 2021). Although plasmonic substrates mediated photoporation can acquire high throughput intracellular delivery, the devices are difficult to apply in vivo.

Figure 2. Overview of photothermal substrates used for nanosecond laser induced photoporation. (A) Scanning electron microscope (SEM) images of the pyramids substrate. Adapted with permission from (Saklayen et al. 2017). Copyright (2017) American Chemical Society. (B) Finite element method simulation showing the temperature of the thermoplasmonic pyramids. Adapted with permission from (Saklayen et al. 2017). Copyright (2017) American Chemical Society. (C) SEM image of the nanocavities substrate. Adapted with permission from (Madrid et al. 2018). Copyright (2018) American Chemical Society. (D) Tilted SEM image of nanocavities substrate. Adapted with permission from (Madrid et al. 2018). Copyright (2018) American Chemical Society. (E) SEM image of fabricated substrate with microwells and sharp tips. Adapted with permission from (Man et al. 2019). Copyright (2019) American Chemical Society. (F) High magnification image of the black square area in (E). Adapted with permission from (Man et al. 2019). Copyright (2019) American Chemical Society.

In addition, there is another way of perforation which is microfluidics mediated photoporation (Breunig et al., 2014; Kaladharan et al., 2021). It was used to deliver foreign cargo automatically. However, the laser source used in reported work is FS laser. It may be interesting to study microfluidics mediated photoporation induced by NS laser.

4. The factors affecting the delivery efficiency of photoporation

For photoporation technique, high delivery efficiency and cell viability are the goals that all studies pursuing. However, there are still many factors restricted the delivery efficiency and cell viability, such as laser energy, number of laser pulses, molecular weight of foreign material, concentration of nanoparticle, nanoparticles type, and cell culture medium.

Laser energy density is a critical factor, as it determines that perforation of cell membrane is caused by heat or VNBs. VNBs generation threshold serves as the dividing line between heat and VNBs. Our previous researches have presented an approach to detect NS laser induced VNBs and have obtained the threshold of VNBs generation (Fu et al., 2018; Wang et al., 2018; 2018). It could provide a reference for laser energy density of photoporation. When laser energy density is below the threshold of VNBs generation, the permeability of cell membrane is changed by laser induced heating. On the contrary, pore will be formed in cell membrane when the laser energy density is strong enough to generate VNBs. Xiong et al. demonstrated that the threshold of VNBs generation was 2.04 J/cm² (Xiong et al., 2014). The delivery efficiency reached to 90% at 2.04 J/cm², while it was much lower at 0.38 J/cm², at around 40%. It demonstrated VNBs induced drug delivery was more efficient than laser induced heating. Baumgart et al. also showed that laser energy density was closely related with delivery efficiency (Baumgart et al., 2012). In addition, the number of laser pulses can also affect delivery efficiency (Liu et al., 2018; Xiong et al., 2021).

The delivery efficiency of nanoparticle-mediated photoporation is also dependent on nanoparticle concentration. Some studies have shown that delivery efficiency increases with the increase of nanoparticles concentration, but the cell viability starts to decline beyond a certain threshold of nanoparticle concentration (Harizaj et al., 2021a; 2021b; 2021c). Therefore, some methods, such as scanning electron microscope, confocal reflection microscopy, reflected light microscopy, and fluorescence-lifetime imaging, were used to quantify the number of nanoparticle attached on cell membrane (Patskovsky et al., 2015; Yao et al., 2017; Raes et al., 2019; Van Hoecke et al., 2019; Patskovsky et al., 2020). The number of nanoparticles attached on cell membrane can affect threshold of VNBs generation, which decreases with the increase of nanoparticle concentration (Wang et al., 2018). Hence, the greater the number of nanoparticles attached to cell membrane, the lower the threshold for VNBs generation becomes. Thus, low laser energy density can be chosen to obtain high delivery efficiency, meanwhile guarantee the cell viability. In addition, some studies have employed different types of nanoparticles to mediate photoporation (Liu et al., 2018; Pylaev et al., 2019; Shaabani et al., 2021; Wang et al., 2021). However, there has not yet been any research to compare the difference of delivery efficiency among these nanoparticles.

Molecular weight of foreign substances is another factor. Several researches showed that the delivery efficiency decreased with increasing molecular weight of foreign materials (Xiong et al., 2016; Raes et al., 2019; Liu et al., 2020; Du et al., 2021). This phenomenon could attribute to diffusion velocity (Liu et al., 2020). Large cargo diffuses slower than small cargo, so the probability of small cargo diffusing into cell is higher than that of large cargo within a certain opening time. Another reason may ascribe to steric hindrance (Liu et al., 2020). Therefore, in order to improve the delivery efficiency, the pore should be large enough and the opening time should be long enough.

In addition, the concentration of foreign materials, incubation time of nanoparticle, cell environment (in PBS or culture medium) and cell state (adherent or suspension) can also affect delivery efficiency (Kalies et al., 2014; Yao et al., 2017). Therefore, photoporation is a complex process affected by many factors. The optimal experimental parameter is a vital issue for pursuing high delivery efficiency and cell viability. Before experiments, each factor should be taken into consideration to balance delivery efficiency and cell viability.

5. Prospects and conclusions

Over the past decades, photoporation technique has been widely studied to deliver the membrane-impermeable foreign materials into living cells. It has been demonstrated to be able to deliver various cargos, however, the delivery efficiency of large cargos still requires improvement. The delivery efficiency is affected by many factors such as laser energy, number of laser pulses, molecular weight, concentration of nanoparticle, nanoparticles type and cell culture medium. These parameters ultimately lead to the factors, pore size and opening time, closely related to delivery efficiency. Pore size acts as a threshold that determines whether the cargo can enter into cell through the pore. The opening time decides the duration for which the cargo can diffuse into cell through the pore. In addition, pore size has a closely relationship with cell viability. Oversize pore may not reseal and lead to cell death. Therefore, it is necessary to detect the pore size and opening time for understanding photoporation mechanism.

For NS laser induced photoporation, there are still some obstacles preventing it to clinic application (Table 2). Low efficiency is a limitation for direct photoporation, but nanoparticles may be a good mediator to enhance loading efficiency. Even so, the toxicity of commonly used nanoparticles is a concern for future clinical application. Therefore, future studies should focus the view on biocompatible and biodegradable materials. In a recent report, Harizaj et al. firstly synthesized a biodegradable polymeric nanoparticle to assist photoporation (Harizaj et al., 2021c). This research made a first attempt to study biodegradable nanoparticle-mediated photoporation. Plasmonic substrates are difficult used in vivo for plasmonic substrates mediated photoporation. Biodegradable nanoparticles combined with a wide-field light source may be a good strategy to achieve high-throughput photoporation instead of the plasmonic substrates.

Table 2. Obstacles and strategies for different modes of photoporation.

Photoporation Type	Obstacles	Strategies	Reference
Direct photoporation	Low efficiency	Nanoparticle enhancement	Shinde et al., 2021
Nanoparticle mediated photoporation	Toxicity of nanoparticle	Biodegradable nanoparticle	Harizaj et al., 2021c
Plasmonic substrates mediated photoporaiton	The substrates are difficult used in vivo	Biodegradable nanoparticle	Harizaj et al., 2021c

Laser beam has a merit that can be easily tuned according to requirement. Wavefront shaping technique has been widely applied in the field of biomedical optics (Yu et al., 2022). It may be a promising research direction to combine photoporation and wavefront shaping technique, which can be used to selectively transfect the targeting cells without affecting surrounding cells. Xiong et al. reported a work to selectively deliver foreign cargo into live cells using a microscope xy-translation stage (Xiong et al., 2017). It may be convenient to accomplish selective delivery by wavefront shaping technique.

Although photoporation technique has been widely reported, it is still challenging to apply photoporation for in vivo intracellular delivery. Because the tissue penetration depth limits its application. However, it may be an opportunity to treat the ophthalmic and dermatological diseases. Laser beam can directly treat skin and eye without attenuation. Moreover, laser technique has already been used for treating skin and ophthalmic pathologies, such as correction of myopia and acne. Sauvage has already reported a work to safely destruct vitreous opacities by laser-induced nanobubbles (Sauvage et al., 2022). Therefore, VNBs mediated photoporation may be a promising tool for treatment of skin and ophthalmic pathologies.

There are still some issues should be concerned for photoporation system. For NS laser, the pulsed energy is instability that may affect the delivery efficiency and cell viability. The repetition frequency may affect the processing speed. Therefore, NS laser with high repetition frequency should be chose to transfect cells.

In conclusion, NS laser induced photoporation has already been studied to resolve some clinical problems, such as antibiotic resistance, vitreous opacities, etc. Compared with FS and CW laser, NS laser is an ideal chose for photoporation considering the price and throughput. Thus, this review has discussed NS laser induced photoporation technique. The mechanism of photoporation induced by different laser has been discussed. The possible factors that may cause changes in delivery efficiency and cell viability have been highlight. Therefore, optimal experimental parameters should be obtained before photoporation. Changes of these factors will result in different pore size and opening time, which directly affect delivery efficiency and cell viability. It may be meaningful to detect the pore size and opening time. Looking into the future, the possible area of photoporation and the drawbacks of the system have been presented. Photoporation technique may have potential applications in clinical settings for skin and ophthalmic pathologies. In summary, NS laser induced photoporation may be an alternative method for solving clinical problems of skin and ophthalmic pathologies.

Ethical approval

No animals or humans were involved in this work and hence no need of ethical approval.

Author contributions

Conceptualization, Rongqian Wu and Cuiping Yao; Writing-original draft preparation, Xiaofan Du, Meng Zhao, Le Jiang, Lihui Pang; Writing-review and editing, Xiaofan Du, Meng Zhao, Jing Wang and Yi Lv; Funding acquisition, Rongqian Wu. All authors have read and agreed to the published version of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This research was supported by Key Research and Development Program of Shaanxi [2022ZDLSF04-09]. The Innovation Capacity Support Plan of Shaanxi Province [2020TD-040]. Institutional Foundation of The First Affiliated Hospital of Xi’an Jiaotong University [2021ZYTS-15].