Rg3, levobupivacaine hydrochloride, and ethanol were obtained from Aladdin. PL-100M and cholesterol were purchased from AVT (Shanghai) Pharmaceutical Tech Co. Ltd. Triethanolamine, bis [3-(triethoxysilyl)propyl] tetrasulfide, cetyltrimethylammonium chloride, as well as tetraethyl orthosilicate, were all acquired from Sigma-Aldrich (United States). Endothelial cell medium was supplied by Sciencell Corporation. The standard CCK-8 assay kit was acquired from Dojindo Molecular Biotechnologies Co., Ltd (Japan). Dialysis bags featuring a 3,500 Da molecular weight cutoff were supplied by Yuanye Biotechnology Co., Ltd.

Transmission electron microscopy images of MSN, MSN-bupi, and LMSN-bupi were acquired using a Hitachi H-7650 instrument, which was operated at 50 kV. Dynamic Light Scattering (DLS) measurements were performed in distilled water utilizing the Litesizer500 instrument (Anton Paar) to ascertain precise particle size distributions. Nitrogen adsorption-desorption isotherms were acquired utilizing a Micromeritics ASAP 2460 analyzer to assess the pore size distribution and determine the Brunauer-Emmett-Teller (BET) specific surface area subsequent to degassing the samples at 120°C for a duration of 6 h. The absorption spectra spanning the ultraviolet-visible-near infrared (UV-vis-NIR) range were precisely recorded utilizing a Shimadzu UV-1900i spectrophotometer.

MSN were synthesized adhering to a previously documented protocol (Wu et al., 2015). Specifically, 2.0 g of CTAC and a suitable quantity of triethanolamine (TEA) were dissolved in 20 mL of distilled water while stirring at 95°C. After a period of 20 min, 1.0 g of TEOS and 1.3 g of BTES were gradually introduced to the mixture, which was then stirred for an additional 4 h. The subsequent product underwent rigorous washing with ethanol and water to eliminate residual reactants. To remove CTAC, the product was refluxed in a 10% (v/v) hydrochloric acid solution in ethanol at 78°C for a duration of 12 h.

A quantity of 10 mg of MSN was dispersed in 5 mL of distilled water. Following this, 10 mg of levobupivacaine was incorporated into the suspension, and the mixture was stirred at room temperature overnight. The suspension was centrifuged at 12,000 rpm for 10 min, and the resultant precipitate was collected, designated as MSN-bupi.

The phospholipid PL-100M and Rg3 (alternatively, cholesterol for conventional liposomes) were intimately blended at a precise weight ratio of 10:3 and subsequently dissolved in chloroform. The solvent was then subjected to vacuum evaporation at a controlled temperature of 48°C, resulting in the formation of a delicate lipid film. This film was subsequently hydrated with PBS solution, ultimately yielding the desired liposomes.

The encapsulation of MSN-bupi nanoparticles within liposomes was achieved through an extrusion method employing a mini-extruder sourced from Avanti Polar Lipids, United States (Xia et al., 2022) Initially, 0.9 mg of pre-fabricated MSN-bupi nanoparticles underwent extensive rinsing with distilled water and were then dispersed in 3 mL of ultrapure water. Concurrently, 0.9 mg of liposome membranes were solubilized in 400 μL of ultrapure water and introduced into the MSN-bupi dispersion. This mixture was then subjected to 11 cycles of extrusion through an 800 nm porous filter membrane using the Avanti mini-extruder. Following this, the resultant dispersion was centrifuged at 12,000 rpm for 10 min at 4°C, and subsequently rinsed three more times with chilled distilled water. The supernatant was then collected for UV-vis spectroscopic analysis to determine the concentration of levobupivacaine. The loading efficiency of levobupivacaine was calculated using the formula: Loading Efficiency = (1 − [levobupivacaine in supernatant]/[total levobupivacaine]) × 100%.

To assess the release characteristics of levobupivacaine from MSN-bupi, LMSN-bupi, and TMSN-bupi nanoparticles, a concentration of 5 mg/mL was encapsulated within dialysis bags featuring a molecular weight cutoff of 3.5 kDa. These bags were then submerged in 20 mL of distilled water, with the entire system maintained at a stable temperature and agitated at 160 rpm utilizing a JINGHONG shaker. At predetermined time points, 5 mL aliquots of the release medium were extracted for levobupivacaine quantification, and an equivalent volume of fresh distilled water was replenished to sustain sink conditions. The cumulative release was subsequently calculated using standard calibration curves derived from UV-vis absorbance measurements.

Using the standardized CCK-8 assay, we evaluated the cytotoxicity of LMSN-bupi and TMSN-bupi on Dorsal Root Ganglion (DRG) cells sourced from BIOSPECIES. Initially, DRG cells were seeded into 96-well plates and cultured overnight. Following the removal of the culture medium, the cells were exposed to varying concentrations of LMSN-bupi and TMSN-bupi in ECM medium and incubated for durations of 12 or 24 h. At the predetermined time points, the medium was substituted with 100 μL of a freshly prepared 10% CCK-8 solution diluted in fresh medium, and the cells were further incubated for an additional 1–2 h. Cell viability was subsequently quantified by recording the absorbance at 450 nm utilizing a microplate reader (Varioskan LUX, Thermo Scientific Inc., United States).

Male Balb/c mice, aged between 4 and 6 weeks, were procured from Liaoning Changsheng Biotechnology Corporation. The Animal Care Committee of Dalian Medical University granted approval for all animal experiments, which were conducted strictly adhering to the institutional guidelines. Prior to undergoing behavioral testing, the mice were housed in a temperature-regulated environment and allowed to acclimate for a period of 2 weeks.

A previously described model for assessing pain in mice through incision was established (Pogatzki and Raja, 2003). Mice were anesthetized with 2% isoflurane. Prior to surgery, the surgical area was thoroughly disinfected using a 10% povidone-iodine solution. A 2 mm longitudinal incision was precisely made, initiating from the proximal edge of the heel. The underlying muscle tissue was gently lifted and incised along its entire length. Subsequently, the skin incision was meticulously closed using 4–0 sutures, and an antibiotic ointment was applied to the wound site to prevent potential infection.

Mice were randomly assigned to groups and received a single local injection (100 μL) around the sciatic nerve (n = 5 per group). The treatments included MSN-bupi suspension, LMSN-bupi suspension, TMSN-bupi suspension, levobupivacaine solution (containing 100 μg of levobupivacaine), or PBS as a control.

Mechanical allodynia was assessed utilizing the von Frey filament test (KW-CT, Kaerwen Inc., China) to evaluate the response to non-noxious mechanical stimuli (Mohd Isa et al., 2018). Mice were positioned on a wire mesh floor within transparent enclosures and given at least 30 min to acclimatize. A probe was gently applied to the plantar surface of the hind paw, incrementally increasing the force applied until a withdrawal response was elicited. The force level at which the withdrawal occurred was documented as the paw withdrawal threshold (PWT). Each mouse underwent a series of 5 trials, and the mean PWT was subsequently calculated for statistical analysis.

Thermal hyperalgesia was evaluated utilizing a hot plate test apparatus (KW-LB, Kaerwen Inc.) maintained at a temperature of 55°C (Wang et al., 2022). Mice were individually placed on the hot plate, and the paw withdrawal latency (PWL)-the time taken for the injured hind paw to be lifted-was automatically recorded. Three measurements were taken for each mouse with intervals of 5–10 min to prevent tissue damage, and a cutoff time of 20 s was established. The average PWL was used for statistical analysis.

Mice underwent euthanasia at 3 and 7 days post-treatment with LMSN-bupi and TMSN-bupi. Subsequently, the sciatic nerve and adjacent tissues were excised and immersed in 4% paraformaldehyde overnight for fixation. These tissues underwent hematoxylin and eosin (H&E) staining, while additional sciatic nerve sections were specifically stained with toluidine blue. An automated digital slide scanner from KFBIO Inc. facilitated the analysis of the stained sections. Furthermore, tissue samples were procured for cytokine assays.

Statistical analyses were performed using GraphPad Prism 9 software. Data are presented as mean ± standard deviation. Comparisons between two groups were made using Student’s t-test, while one-way ANOVA was used for multiple group comparisons. A p-value of less than 0.05 was considered statistically significant.

Rg3-based liposomes and conventional liposomes were prepared using the thin-film hydration method as previously described. Dendritic mesoporous silica nanoparticles (MSN) were synthesized following established protocols. Transmission electron microscopy (TEM) images confirmed that the synthesized MSN exhibited a spherical morphology with large pores (Figure 1A). To enhance pain management capabilities, levobupivacaine hydrochloride was loaded into MSN to form MSN-bupi nanoparticles. TEM images showed increased opacity within the particles after levobupivacaine loading, indicating that the inner pores were filled (Figure 1B). To fabricate liposome-camouflaged MSN-bupi (LMSN-bupi) incorporating Rg3, a blend of Rg3-based liposomes and MSN-bupi was extruded through a mini-extruder for a total of 11 passes. TEM images of the resulting LMSN-bupi nanoparticles displayed spherical core–shell structures, confirming successful coating with Rg3-based liposomes (Figure 1C) (Qiao et al., 2020) Dynamic light scattering (DLS) analysis revealed an increase in hydrodynamic diameter for LMSN-bupi (280 nm, PDI 0.256) compared to uncoated MSN (247 nm, PDI 0.318) and MSN-bupi (259 nm, PDI 0.197), indicating successful liposome coating (Figures 1D–F). The zeta potential shifted from −42.03 ± 1.7 5 mV for MSN to approximately −18.33 ± 6.59 mV after loading with levobupivacaine to form MSN-bupi, and further to −6.6 ± 2.1 mV upon coating with Rg3-based liposomes to produce LMSN-bupi (Figure 1G). Nitrogen adsorption-desorption isotherms were measured to confirm the mesoporous structure of MSN (Figures 1H, I). The hysteresis loops observed are characteristic of mesoporous materials. The Brunauer-Emmett-Teller (BET) surface area (553 mm²/g) and average pore diameter (18.3 nm) indicated a high porosity of the MSN. Based on the UV-vis-NIR absorption spectra analysis, the encapsulation efficiency of levobupivacaine within the MSN-bupi matrix was precisely determined to be 86.4%.

The liposome camouflaging of the nanoparticles may have contributed to plugging the pores of the MSN, thereby enhancing the sustained release of levobupivacaine. To evaluate this effect, levobupivacaine release profiles from different nanoparticles were observed at predetermined time points. Notably, the differences in levobupivacaine release between LMSN-bupi and MSN-bupi highlight the impact of liposome decoration (Figure 2A). Under identical conditions, LMSN-bupi exhibited a slower release of levobupivacaine compared to MSN-bupi. Importantly, no significant variation was observed between LMSN-bupi and TMSN-bupi, indicating that the drug release process was primarily governed by the liposome coating rather than the specific liposome composition. At specific time points, the cumulative release of levobupivacaine from the MSN-bupi group was significantly higher than that from the LMSN-bupi and TMSN-bupi groups (Figure 2B). This sustained release characteristic of LMSN-bupi nanoparticles is advantageous for prolonged pain management.

The cytotoxicity of LMSN-bupi and TMSN-bupi toward dorsal root ganglion (DRG) cells was assessed using the standard CCK-8 assay (Liang et al., 2022). DRG cells were chosen due to their representative nature of normal cells that play a crucial role in the transmission of pain. After a 24-hour co-incubation period with LMSN-bupi and TMSN-bupi, the results of the cell viability assays revealed no statistically significant difference between the treated and control groups, suggesting that these nanoparticles had no detrimental effect on the growth of DRG cells (Figure 3A). Furthermore, DRG cells co-incubated with LMSN-bupi for 12 h and 24 h exhibited unchanged viability (Figure 3B), corroborating the high biocompatibility of LMSN-bupi for pain management applications.

The efficacy of LMSN-bupi nanoparticles for pain management was evaluated in vivo using a mouse incision pain model. Three hours following surgery, a significant decrease in mechanical threshold and thermal latency was observed, confirming the successful induction of pain. Subsequently, the mice underwent perineural injection of levobupivacaine, TMSN-bupi, or LMSN-bupi in the vicinity of the sciatic nerve. To assess mechanical hyperalgesia, behavioral tests were conducted utilizing mechanical stimulation devices that precisely measured the mechanical threshold (Figures 4A, B). Following injection, thermal and mechanical pain responses were meticulously monitored at 2-hour intervals. Notably, the injection of 100 μL of PBS (serving as the control) did not elicit any changes in the mechanical threshold (Figure 4A). Conversely, the administration of 100 μL of levobupivacaine effectively produced a pain-blocking effect that persisted for approximately 6 h. In contrast, administration of 100 μL of TMSN-bupi extended the duration of analgesia to about 12 h, highlighting the potential of nanotechnology to prolong the effect of local anesthetics. Notably, administration of 100 μL of LMSN-bupi further extended the analgesic duration up to 18 h compared to TMSN-bupi, which can be attributed to the intrinsic anti-inflammatory properties of Rg3. Mice receiving PBS, TMSN, or LMSN without levobupivacaine showed no analgesic effect, indicating that MSN alone did not contribute to pain management (Figure 4B).

Paw Withdrawal Latency (PWL) under thermal stimulation was evaluated to assess thermal hyperalgesia. Injection of PBS did not alter the thermal response (Figure 4C), whereas the administration of levobupivacaine induced a 6-hour pain blockade. Likewise, mice subjected to sciatic nerve injection with TMSN-bupi displayed a prolonged PWL on a 55°C hot plate for approximately 12 h, in comparison to levobupivacaine administration alone, indicative of enhanced pain management. Notably, LMSN-bupi exhibited a markedly longer duration of analgesia than TMSN-bupi, which can be attributed to the Rg3-based liposome modification that prolongs in vivo retention time. In contrast, negligible analgesic effects were observed in the PBS, TMSN, and LMSN groups devoid of levobupivacaine, highlighting that the nanoparticles alone did not contribute to pain alleviation (Figure 4D). By 48 h, both thermal and mechanical thresholds in all groups had reverted to baseline levels, verifying the temporary nature of the analgesic effects and validating the incision pain model.

To evaluate the biosafety of LMSN-bupi in managing pain, mice that received LMSN-bupi injections were euthanized 7°days post-administration. The sciatic nerve, along with adjacent muscle and skin tissues, were collected and underwent hematoxylin and eosin (H&E) staining as well as toluidine blue staining (Bai et al., 2024). Histological analysis indicated an absence of swelling, discoloration, or tissue damage (Figure 5). Recognizing the potential limitations of H&E staining in detecting nerve injury, toluidine blue staining was additionally employed on sciatic nerve sections. Toluidine blue specifically stains Nissl bodies, serving as a marker of neuronal integrity. After 7 days, no notable peripheral nerve damage was detected. These findings underscore the exceptional biocompatibility of LMSN-bupi nanoparticles in the context of pain therapy.

To examine the impact of LMSN-bupi on inflammatory cytokines during pain management, mice injected with varying nanoparticles were euthanized at 3 and 7°days post-injection. The sciatic nerve, along with adjacent muscle and skin tissues, were harvested for a detailed cytokine analysis using ELISA. When compared to the control group, mice administered with TMSN-bupi displayed heightened concentrations of pro-inflammatory cytokines, including IL-1β, IFN-γ, and TNF-α, indicative of a potential inflammatory response induced by TMSN-bupi (Figures 6A–F) (Wu et al., 2023; Jiang et al., 2024) In contrast, mice treated with LMSN-bupi showed reduced cytokine levels at both 3 and 7°days post-injection, which can be attributed to the anti-inflammatory effects of Rg3. These findings demonstrate that LMSN-bupi nanoparticles not only provide effective pain relief but also modulate inflammatory responses, enhancing the overall efficacy of pain management.