Dual-responsive PDA-HP hydrogel enables mitochondria-targeted mild photothermal therapy for spinal cord repair methods
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Design and preparation of PDA-HP hydrogel
PDA nanoparticles were synthesized via alkaline oxidative self-polymerization [ ]. Briefly, a mixture of 40 mL absolute ethanol, 90 mL deionized water, and 2 mL ammonium hydroxide (28-30 %) was magnetically stirred in a round-bottom flask. Dopamine hydrochloride (250 mg) dissolved in 10 mL deionized water, was then added, and the reaction was allowed to proceed for 24 h at room temperature. The resulting PDA nanoparticles (average diameter ≈ 193 nm) were collected by centrifugation (15,000 rpm, 10 min), thoroughly washed, and lyophilized.
Design and preparation of PDA-HP hydrogel
Heparin-poloxamer (HP) conjugates were prepared via EDC/NHS-mediated coupling. Poloxamer 407 was first activated with 1.3 mM 4-nitrophenyl chloroformate, followed by reaction with ethylenediamine to produce an amine-terminated intermediate. This intermediate was subsequently reacted with heparin in MES buffer overnight at room temperature. The resulting HP conjugate was purified by extensive dialysis over 3 days and then lyophilized to yield a dry powder.
Determination of steady-state viscosity of HP hydrogel and PDA-HP hydrogel
HP hydrogel and PDA-HP hydrogel samples (60 mL each) were equilibrated in a thermostatic bath with a temperature control precision of ±0.1 °C. A digital rotational viscometer (NDJ-1S, Shanghai, China) equipped with a T-bar guard and spindle No. 1 was used. The rotor was lowered until the engraved line aligned with the meniscus of the sample. The temperature was increased from 20 °C to 50 °C in increments of 5-7 °C, fully covering the mild photothermal range (37-43 °C) relevant to in vivo PTT conditions. After each 10-min equilibration period, viscosity measurements were recorded at 30 rpm. Each measurement was performed in triplicate, and the mean values were used for statistical analysis.
Photothermal performance measurement
Temperature changes were continuously recorded using both an infrared thermal camera (Fluke TiX560, USA) and a K-type thermocouple probe positioned at the center of the hydrated sample to verify the accuracy of surface and internal temperature measurements. Each heating-cooling cycle was conducted under identical irradiation parameters (808 nm, 1.0 W/cm 2, 10 min per cycle), followed by natural cooling to room temperature before the next cycle, ensuring consistent thermal exposure across all tests.
Photothermal performance measurement
Heating-cooling cycles were repeated six times to evaluate photothermal stability, and temperature-time curves were plotted to assess concentration-dependent heating behavior. For in-vivo thermographic monitoring, infrared imaging was performed at the spinal lesion site during irradiation. The surface temperature was maintained below 43 °C, consistent with the definition of mild photothermal therapy, and no visible tissue damage was observed.
Photothermal treatment and hydrogel co-culture
For co-culture experiments, ND7/23 cells were seeded in 24-well plates (1 × 10 5 cells/well) and incubated with 100 µL of PDA-HP pre-gel solution per well. The mixture was maintained at 37 °C for 10 min to allow complete gelation, forming a thin hydrogel layer over the cells. After 24 h of incubation, the cultures were irradiated with an 808 nm NIR laser (1 W/cm 2 ) for 10 min at a fixed distance of 5 cm. The temperature of the culture medium was continuously monitored with a thermocouple and remained below 43 °C to ensure mild photothermal treatment. Control groups were handled identically without laser exposure.
Animal model of SCI
All experimental procedures were approved by the Institutional Review Board of Gannan Medical University. C57BL/6 mice were purchased from SLAC and housed under controlled conditions at 22-26 °C with 35-55 % relative humidity, with free access to food and water. Female C57BL/6 mice (8 weeks old, 20-22 g) were used in all experiments. Following complete depilation and sequential disinfection of the surgical site with povidone-iodine and 70 % ethanol, general anesthesia was induced with 5 % isoflurane in 1 l/min O 2 and maintained at 2 %. Body temperature was continuously monitored and maintained at 37 °C on a heated surgical platform, and adequate anesthesia was confirmed by the absence of a tail-pinch reflex. A midline skin incision was made, and the paravertebral muscles were bluntly dissected to expose the T9-T10 ve...
Histological staining
For comprehensive histological evaluation, spinal cord, heart, liver, spleen, lung, kidney, and knee-joint (femur-tibia) specimens were fixed in 4 % paraformaldehyde at 4 °C for 48 h. Bones were decalcified in 10 % EDTA (pH 7.4) for 14 days, followed by dehydration, paraffin embedding, and sagittal sectioning at 5 µm. Sections were deparaffinized, rehydrated, and stained with hematoxylin for 5 min and eosin for 2 min.
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PDA nanoparticles were synthesized via alkaline oxidative self-polymerization [ ]. Briefly, a mixture of 40 mL absolute ethanol, 90 mL deionized water, and 2 mL ammonium hydroxide (28-30 %) was magnetically stirred in a round-bottom flask. Dopamine hydrochloride (250 mg) dissolved in 10 mL deionized water, was then added, and the reaction was allowed to proceed for 24 h at room temperature. The resulting PDA nanoparticles (average diameter ≈ 193 nm) were collected by centrifugation (15,000 rpm, 10 min), thoroughly washed, and lyophilized.
Heparin-poloxamer (HP) conjugates were prepared via EDC/NHS-mediated coupling. Poloxamer 407 was first activated with 1.3 mM 4-nitrophenyl chloroformate, followed by reaction with ethylenediamine to produce an amine-terminated intermediate. This intermediate was subsequently reacted with heparin in MES buffer overnight at room temperature. The resulting HP conjugate was purified by extensive dialysis over 3 days and then lyophilized to yield a dry powder.
HP hydrogel and PDA-HP hydrogel samples (60 mL each) were equilibrated in a thermostatic bath with a temperature control precision of ±0.1 °C. A digital rotational viscometer (NDJ-1S, Shanghai, China) equipped with a T-bar guard and spindle No. 1 was used. The rotor was lowered until the engraved line aligned with the meniscus of the sample. The temperature was increased from 20 °C to 50 °C in increments of 5-7 °C, fully covering the mild photothermal range (37-43 °C) relevant to in vivo PTT conditions. After each 10-min equilibration period, viscosity measurements were recorded at 30 rpm. Each measurement was performed in triplicate, and the mean values were used for statistical analysis.
Temperature changes were continuously recorded using both an infrared thermal camera (Fluke TiX560, USA) and a K-type thermocouple probe positioned at the center of the hydrated sample to verify the accuracy of surface and internal temperature measurements. Each heating-cooling cycle was conducted under identical irradiation parameters (808 nm, 1.0 W/cm 2, 10 min per cycle), followed by natural cooling to room temperature before the next cycle, ensuring consistent thermal exposure across all tests.
Heating-cooling cycles were repeated six times to evaluate photothermal stability, and temperature-time curves were plotted to assess concentration-dependent heating behavior. For in-vivo thermographic monitoring, infrared imaging was performed at the spinal lesion site during irradiation. The surface temperature was maintained below 43 °C, consistent with the definition of mild photothermal therapy, and no visible tissue damage was observed.
For co-culture experiments, ND7/23 cells were seeded in 24-well plates (1 × 10 5 cells/well) and incubated with 100 µL of PDA-HP pre-gel solution per well. The mixture was maintained at 37 °C for 10 min to allow complete gelation, forming a thin hydrogel layer over the cells. After 24 h of incubation, the cultures were irradiated with an 808 nm NIR laser (1 W/cm 2 ) for 10 min at a fixed distance of 5 cm. The temperature of the culture medium was continuously monitored with a thermocouple and remained below 43 °C to ensure mild photothermal treatment. Control groups were handled identically without laser exposure.
All experimental procedures were approved by the Institutional Review Board of Gannan Medical University. C57BL/6 mice were purchased from SLAC and housed under controlled conditions at 22-26 °C with 35-55 % relative humidity, with free access to food and water. Female C57BL/6 mice (8 weeks old, 20-22 g) were used in all experiments. Following complete depilation and sequential disinfection of the surgical site with povidone-iodine and 70 % ethanol, general anesthesia was induced with 5 % isoflurane in 1 l/min O 2 and maintained at 2 %. Body temperature was continuously monitored and maintained at 37 °C on a heated surgical platform, and adequate anesthesia was confirmed by the absence of a tail-pinch reflex. A midline skin incision was made, and the paravertebral muscles were bluntly dissected to expose the T9-T10 vertebrae. The T9 spinous process, angled caudally, and the neutral to slightly caudally tilted T10 process served as anatomical landmarks. Following complete laminectomy at T9-T10, the spinal cord was fully exposed. A moderate contusion was induced at the midline using a 10 g stainless-steel rod...
For comprehensive histological evaluation, spinal cord, heart, liver, spleen, lung, kidney, and knee-joint (femur-tibia) specimens were fixed in 4 % paraformaldehyde at 4 °C for 48 h. Bones were decalcified in 10 % EDTA (pH 7.4) for 14 days, followed by dehydration, paraffin embedding, and sagittal sectioning at 5 µm. Sections were deparaffinized, rehydrated, and stained with hematoxylin for 5 min and eosin for 2 min.
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