Optical Tweezers-Assisted siRNA Delivery for Gene Silencing in Plant Protoplasts

1. Introduction

Reducing agriculture’s reliance on chemical pesticides is a pressing imperative for environmental health [1,2]. RNA interference (RNAi) represents an eco-friendly strategy to address this challenge, enabling the development of pest- and pathogen-resistant crops through sequence-specific gene silencing [3,4]. However, broad practical deployment of RNAi is still hindered by a major bottleneck: the low efficiency of delivering small interfering RNA (siRNA) [5]. Conventional physical delivery methods, such as gene gun bombardment, typically achieve only 0.5 - 2% transient delivery efficiency in Nicotiana cells and frequently cause cell mortality exceeding 30% [6]. While nanocarrier systems [7] have improved delivery efficiency to 30 - 70% relying on Brownian-motion-driven diffusion [8,9], their insufficient target specificity and unresolved long-term biosafety concerns limit practical application [10]. These limitations motivate the exploration of efficient and bio-compatible delivery platforms to unlock the capability of RNAi-related technologies.

Optical tweezers, which use a tightly focused laser beam to create a three-dimensional optical trap, allow noncontact, noninvasive and high spatiotemporal-resolution manipulation of micro- and nanoparticles [11,12]. Such capabilities have fostered broad applications in precision measurement [13,14,15], nanotechnology [16,17,18], and biological science [19,20]. In biomedicine, optical tweezers have been adopted for targeted delivery of diverse biomolecular cargoes, such as siRNA, plasmid DNA, proteins, and isolated mitochondria [21,22]. Furthermore, “nano-tweezers” enable noninvasive extraction of single-molecule biomolecules from living cells [23]. Despite the promising prospects of optical tweezers techniques in those fields, using optical tweezers to deliver nucleic acids into plant cells has yet to be experimentally demonstrated. The precise and non-invasive nature of optical tweezers presents a promising alternative to overcome the difficulty of achieving targeted delivery in plant protoplast transfection.

Colloidal microcarriers loaded with biological molecules have been widely used in targeted delivery [24]. However, most existing works mainly focus on delivery performance, while ignoring the nanoscale interfacial stability between the loaded molecules and the microcarriers in optical tweezers [25]. Intense local optical field gradients and photothermal effects in optical tweezers may perturb the nanometer-thin adsorption interface and trigger premature dissociation of adsorbed biomolecules [26,27]. Without evaluation of such interfacial stability, it remains difficult to judge whether the cargo can stay firmly bound to microcarriers through-out the optical manipulation process [28,29,30].

In this work, amino-modified polystyrene (PS) microspheres are synthesized and assembled with siRNA to construct cargo-carrying probes. We investigate the nanoscale interfacial stability of such assemblies using optical tweezers, and establish an optical tweezers-assisted strategy for delivering siRNA into single cells. As shown in Figure 1, the trapped siRNA-PS probes can achieve rapid, controllable and noninvasive gene delivery to Nicotiana protoplasts. This approach avoids physical damage and eliminates the randomness inherent in passive nanocarrier uptake. The reduction in green fluorescent protein fluorescence intensity and mRNA expression levels confirm effective gene-silencing performance. Our work establishes a precise and efficient optical delivery platform, providing experimental support for nanophotonic manipulation of micro-scale colloidal assemblies for single-cell cargo delivery.

2. Methods

Synthesis and characterization of siRNA-PS probes. Nucleic acid probes are fabricated via electrostatic binding between amino-modified blue-fluorescent PS microspheres and CY3-labeled siRNA (Figure 2a). Zeta potential measurements and confocal laser scanning microscopy (CLSM) are employed to evaluate binding efficiency and probe fluorescence properties. Overlapping fluorescence signals in CLSM images (Figure 2b) confirmed successful siRNA adsorption. Zeta potential analysis (Figure 2c) revealed surface charge inversion. The potential shifted from +6.17 mV to -20.7 mV after siRNA loading, further verifying siRNA-PS probe formation. Dynamic light scattering (DLS) is used to assess whether siRNA loading triggers particle aggregation or alters monodispersity. As shown in Figure 2d, the hydrodynamic diameter only marginally increased from 1073.77 nm to 1082.53 nm upon siRNA modification, demonstrating negligible aggregation.

To find the optimal loading ratio of siRNA to PS microspheres, we conduct gel electrophoresis [31], designing the mass ratio of siRNA to PS ranging from 1:1 to 1:50. When the ratio of siRNA to PS is inappropriate, bands appear in the gel. As shown in Figure 2e, the siRNA bands disappear in the range of 1:15 to 1:30 mass ratios, indicating that the siRNA reaches a saturated loading state on the PS micro-spheres. To maximize the amount of siRNA loaded, we select the 1:15 ratio for subsequent experiments. The amount of siRNA in our probe is 40000-fold higher than that of the nanocarrier at a single microsphere level [32], as shown in Figure 2f.

3. Results and Discussion

Stability of siRNA-PS Probes. We characterize the siRNA-PS probes using transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDX). TEM images in Figure 2a reveal no morphological damage to PS micro-spheres after siRNA loading. Since PS microspheres are phosphorus-free, the presence of phosphorus clearly indicates siRNA loading. EDX analysis demonstrates uniform phosphorus distribution across the microsphere surface, confirming homogeneous siRNA loading, as shown in Figure 3a and Table 1.

We analyzed the effects of short-term storage, temperature fluctuations, and enzymatic degradation on the stability of siRNA-PS probes. As shown in Figure 3b, after the probes are stored at room temperature (25 ℃) for 12 hours, no significant siRNA release is observed, indi-cating their short-term stability. Then, the probes demonstrate excellent thermal stability, with considerable siRNA release occurring only at temperatures exceeding 35 ℃, as shown in Figure 3c. This suggests that the probes remain stable under typical plant physiological conditions and during standard laboratory handling. Furthermore, an effective delivery system must protect nucleic acids from enzymatic degradation in the environment [33]. As illustrated in Figure 3d, free siRNA is rapidly degraded by nucleases, whereas siRNA encapsulated within the PS microspheres remains predominantly intact under the same conditions. This confirms the PS microspheres’ protective function and highlights their importance in maintaining siRNA stability for successful delivery into plant cell.

Optical Tweezers-Assisted Delivery. We use optical tweezers to trap and deliver siRNA-PS probes, as shown in Figure 4a. The system is constructed around an inverted microscope. Key components include a high numerical aperture oil-immersion objective (100×, NA = 1.25) , which tightly focuses the trapping laser to form stable optical traps, and a near-infrared semiconductor laser (980 nm, 100 mW) providing a linearly polarized, fundamental-mode Gaussian beam [34,35]. Functionalized PS microspheres (radius = 0.5 μm) serve as siRNA delivery probes. The tightly focused laser beam traps and manipulates the microspheres, with their displacement controlled by a piezoelectric platform [36,37]. A 10× objective lens and a charge-coupled device (CCD) camera are used for real-time observation, while a quadrant photodetector (QPD) detects the forward-scattered signal from the probe [38,39]. We measured the confined Brownian motion and calculated the transverse optical trap stiffness using the power spectrum method, as shown in Figure 4b,c. The results show that the trapping stiffness in the X and Y directions is 3.2 and 3.0 pN/μm/mW, respectively.

To evaluate whether the local photothermal effect at the center of the optical trap causes siRNA to dissociate from the surface of the PS microspheres, we first trap a siRNA-PS probe and observe it for 10 minutes. At this time, the temperature at the center of the light trap is 25.3 ℃. Subsequently, we place the trapping chamber loaded with the probe under CLSM for observation. The results in Figure 4d show that the red fluorescent siRNA remains bound to the surface of the PS micro-sphere and does not appear in the surrounding medium. This indicates that the local photothermal effect at the center of the optical trap does not affect the stability of the probe on the surface of the microspheres.

As shown in Figure 5a, we deliver siRNA-PS to the plant protoplasts using optical tweezers within 12 s. Conventional nanocarriers rely on random Brownian motion for cell contact and internalization, requiring approximately 1 h for siRNA delivery [40,41]. Our approach reduces delivery time by two orders of magnitude, thereby improving delivery efficiency. As shown in Figure 5b and Figure 5c, We design three paths (linear, L-shaped, and π-shaped) for delivering siRNA–PS probes to the plant protoplasts [42]. The delivery times are 10 s, further demonstrating the targeting and high efficiency of our optical tweezers-assisted delivery method.

Gene silence. In this experiment, the gene silencing effect is evaluated using CLSM and quantitative reverse-transcription PCR (RT-qPCR). Green fluorescent protein (GFP) is used as a fluorescent reporter. GFP can produce visible green fluorescence inside cells, and weaker fluorescence signals mean stronger inhibition of target gene expression [43]. CLSM qualitatively assessed gene silencing by monitoring fluorescence changes of GFP protein, whereas RT-qPCR provides quantitative analysis of intracellular GFP messenger-RNA (mRNA) expression levels. mRNA carries gene information inside cells, and lower mRNA content corresponds to better gene silencing performance.

All experimental data are statistically analyzed and expressed as means. Stu-dent’s t-test for two-group comparisons and one-way analysis of variance (ANOVA) for multiple-group comparisons are adopted to judge whether the numerical difference between different cell groups comes from experimental treatment or random test error. The P value is a probability index calculated by statistical methods to measure the reliability of group differences. Data analysis and graphing are per-formed using GraphPad Prism 9.0. Results are considered statistically significant at a P value < 0.05, which means the observed difference is not caused by accidental measurement fluctuations.

Nicotiana protoplasts carrying the GFP gene are adopted for this test. Six treatment groups are designed to evaluate the silencing performance of optical tweezers-assisted delivery (Figure 6a). Untreated protoplasts serve as the blank control (CK group). The PS group containing only PS microspheres verifies that the carrier itself cannot trigger gene silencing. The Ns-S group with PS microspheres and non-specific siRNA tests the specificity of RNA interference. The free siRNA group confirms the inherent silencing capacity of siRNA molecules. The siR-NA-PS-R group represents conventional passive delivery relying on Brownian diffu-sion of siRNA-loaded microspheres. The siRNA-PS-OT group corresponds to siR-NA-PS probes delivered with optical tweezers.

Figure 6b and Figure 6c respectively display fluorescence images and corresponding fluorescence intensities for all experimental groups. Strong GFP fluorescence intensity is observed in the CK, PS, and Ns-PS groups, which proves that no obvious silencing of the GFP gene occurs within these groups. GFP fluorescence in-tensity drops in the free siRNA group and declines further in the siRNA-PS-R group. The siRNA-PS-OT group exhibits the weakest fluorescence signal. These results in-dicate that only specific siRNAs can achieve gene silencing effectively, and the optical tweezers-assisted delivery method produces the optimal silencing effect.

Gene silencing efficiency is quantified by calculating the relative reduction of GFP mRNA expression in each group compared with the CK group. Figure 6d presents the relative expression level of GFP mRNA. The calculated silencing efficiencies reach 58% for the free siRNA group, 75% for the siRNA-PS-R group and 92% for the siRNA-PS-OT group. Therefore, the delivery strategy based on optical tweezers achieves the the best gene silencing performance.

4. Conclusions

In this study, we proposed an optical tweezers-assisted strategy for targeted siRNA delivery into plant protoplasts to achieve fast siRNA transport and high-efficiency gene silencing at the single-cell scale. This approach reduces delivery time by two orders of magnitude, dropping from around 1 h to under 10 s, and improves gene-silencing efficiency from 75% up to 92%. Traditional nanocarrier delivery relies on random Brownian motion diffusion for cell uptake and lacks real-time monitoring and single-probe manipulation capabilities, whereas our strategy provides precise, controllable cargo delivery.

Sonication and microfluidic-based delivery methods possess limited targeting performance, they rely on batch processing and tend to disrupt cell membranes, threatening cell viability and inducing off-target delivery. Optical tweezers, however, support minimally invasive, high-precise single-probe delivery, which is particularly suitable for fragile plant protoplasts [44,45].

Our optical tweezers-assisted siRNA delivery realizes controllable, site-specific gene regulation. Unlike conventional nanocarrier delivery, which relies on random Brownian motion and lacks single-cell control, this method enables precise manipulation of individual plant cells with minimal invasiveness. The proposed method not only achieves efficient gene silencing but also provides a versatile platform for delivering other biomolecules into plant cells, offering new opportunities for plant functional genomics, crop improvement, and sustainable agriculture.